Means and methods for treating and diagnosing fibrosis or fibrosis-associated diseases

ABSTRACT

The present invention is concerned with a protein oligomer comprising (i) at least two NC-1 monomers of collagen 18 or (ii) at least two endostatin domains of collagen 18 or (iii) at least two N-terminal peptides of the collagen 18 endostatin domain, for use in treating, ameliorating or preventing fibrosis or a fibrosis-associated disease, a vascular endothelial growth factor (VEGF)-related disease or a matrix metalloproteinase (MMP)-related disease.. The invention further relates to the mentioned protein oligomer for use for detecting and/or diagnosing fibrosis or a fibrosis-associated disease, a vascular endothelial growth factor (VEGF)-related disease or a matrix metalloproteinase (MMP)-related disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims priority to U.S. patentapplication Ser. No. 15/781,028 filed on Jun. 1, 2018, which is a U.S.National Phase of International PCT Application No. PCT/EP2016/079777filed on Dec. 5, 2016, which claims priority to European PatentApplication No. 15197746.9 filed on Dec. 3, 2015. The contents of eachapplication are incorporated herein by reference in their entireties.

STATEMENT REGARDING SEQUENCE LISTING

This application incorporates by reference the material in the ST.26 XMLfile titled ASD05-62_Replacement_Sequence_Listing, which was created onAug. 15, 2023 and is 40 KB.

The present invention is concerned with a protein oligomer comprising(i) at least two NC-1 monomers of collagen 18 or (ii) at least twoendostatin domains of collagen 18 or (iii) at least two N-terminalpeptides of the collagen 18 endostatin domain, for use in treating,ameliorating or preventing fibrosis or a fibrosis-associated disease, avascular endothelial growth factor (VEGF)-related disease or a matrixmetalloproteinase (MMP)-related disease. The invention further relatesto the mentioned protein oligomer for use for detecting and/ordiagnosing fibrosis or a fibrosis-associated disease, a vascularendothelial growth factor (VEGF)-related disease or a matrixmetalloproteinase (MMP)-related disease.

Excessive deposition of extracellular matrix (ECM) components such asFibronectin (FN) and type I collagen (Col 1α1) by organ fibroblasts isdefined as fibrosis. Organ fibrosis is the final common pathway for manydiseases that result in end-stage organ failure. Uncontrollablewound-healing responses, including acute and chronic inflammation,angiogenesis, activation of resident cells and extracellular matrixremodeling are thought to be involved in the pathogenesis of fibrosis.

Pulmonary fibrosis comprises a group of interstitial disorders of thelung parenchyma that develop as a consequence of multiple causes,including radiotherapy and chemotherapy for lung neoplasms (Am. J.Respir. Crit Care Med. (2002) 165, p. 277-304; Movsas et al. (1997)Chest 111, p. 1061-1076). The pathophysiologic events induced byradiation have striking similarities to those that occur after othertypes of lung injury, such as surgery, chemotherapy, and idiopathicpulmonary fibrosis (IPF) (Rubin et al. (1995), Int. J. Radiat. Oncol.Biol. Phys. 33, p. 99-109).

In a study published in 2006 based on a United States healthcare claimsdatabase, the prevalence of IPF was between 14-42.7 per 100,000,depending on whether narrow or broad case-finding criteria was used(Raghu et al. (2006), Am. J. Respir. Crit. Care Med. 174, p. 810-6).More recently, in May 2012, a systematic survey of literature estimatedthe prevalence of idiopathic pulmonary fibrosis (IPF) in the EuropeanUnion to be 26 per 100,000. The findings of various studies on theincidence of IPF are summarized in a review by Rafii et al. (J. ThoracDis. (2013), 5, p. 48-73). IPF represents the most common cause of deathfrom progressive lung disease. Retrospective studies suggest that themedian survival after diagnosis of IPF is 2-3 years, however, the courseof IPF is variable, with some patients experiencing long periods ofstability while others have frequent exacerbations or a rapid decline(Raghu et al. (2011), Am. J. Respir. Crit. Care Med. 183, p. 788-824;Selman et al. (2001), Ann. Intern. Med. 134, p. 136-51; Boon et al.(2009), PLoS One, 4, e5134).

Clinically, IPF is characterized by interstitial infiltrates,progressive dyspnea, and worsening of pulmonary function that may leadto death from respiratory failure (Am. J. Respir. Crit Care Med. (2002),165, p. 277-304; Allen and Spiteri (2002), Respir. Res. 3, p. 13; Grossand Hunninghake (2001), N. Engl. J. Med. 345, p. 517-525).

An ideal animal model for IPF does not yet exist, but bleomycin- andradiation-induced lung fibrosis models have been used to study lungfibrosis so far (Rubin, loc. cit.).

From a molecular point of view, TGF-beta (TGF-β) is the prototypefibrotic cytokine which is increased in fibrotic organs and contributesto the development of fibrosis by stimulating the synthesis ofextracellular matrix molecules, activating fibroblasts to a-smoothmuscle actin-expressing myofibroblasts, and down-regulating matrixmetalloproteinases (MMPs). Though aberrant TGF-beta expression isimplicated in the pathogenesis of fibrosis in systemic sclerosis, ananti-TGF-beta monoclonal antibody evaluated in a small trial of earlysystemic sclerosis failed to show any efficacy (Varga et al. (2009),Nature Reviews Rheumatology 5, p. 200-6).

The findings of another study suggested a pivotal role of PDGF signalingin the pathogenesis of pulmonary fibrosis and indicated that inhibitionof fibrogenesis, rather than inflammation, is critical to anti-fibrotictreatment (Abdollahi et al. (2005), J. Exp. Med. 201, p. 925-35).

Recently, anti-fibrotic activity has been reported for C-terminalendostatin polypeptides but not for N-terminal endostatin polypeptides,in TGF-beta- and bleomycin-induced fibrosis (WO 2011/050311; Yamaguchiet al. (2012), Sci. Transl. Med., 4, p. 136ra71). Endostatin is anaturally occurring 183-amino acid proteolytic fragment of collagenXVIII that localizes in the basement membrane around blood vessels. Theanti-tumor properties of this protein have been extensively described,demarcating endostatin as an endogenous inhibitor of angiogenesis[Bergers, G., et al., Effects of angiogenesis inhibitors on multistagecarcinogenesis in mice. Science, 1999. 284(5415): p. 808-12; O'Reilly,M. S., et al., Endostatin: an endogenous inhibitor of angiogenesis andtumor growth. Cell, 1997. 88(2): p. 277-85., Folkman, J.,Antiangiogenesis in cancer therapy—endostatin and its mechanisms ofaction. Exp Cell Res, 2006. 312(5): p. 594-607]. Further, it supressesmany signaling cascades such as pro-inflammatory NF-κB, coagulation andadhesion cascades [Abdollahi, A., et al., Transcriptional networkgoverning the angiogenic switch in human pancreatic cancer. Proc NatlAcad Sci U S A, 2007. 104(31): p. 12890-5].

Despite the medical need, there has been remarkably little progress inthe development of effective therapeutic strategies for fibrosis thusfar (see, e.g., Am. J. Respir. Crit Care Med. (2002), 165, p. 277-304;Allen and Spiteri, loc. cit.; Gross and Hunninghake, loc. cit.; Kamp(2003), Chest 124, p.1187-11909; Mason et al. (1999), Am. J. Respir.Crit. Care Med. 160, p.1771-1777; Rafii et al. (2013), J. Thorac Dis. 5,p. 48-73).

There is, thus, a need in the art for the development of effectivetreatment modalities for fibrosis.

The technical problem underlying the present invention could be seen asthe provision of means and methods which comply with the afore-mentionedneeds. This technical problem has been solved by the embodimentscharacterized in the claims and herein below.

Accordingly, the present invention relates to a protein oligomercomprising (i) at least two NC-1 monomers of collagen 18 or (ii) atleast two endostatin domains of collagen 18 or (iii) at least twoN-terminal peptides of the collagen 18 endostatin domain, for use intreating, ameliorating or preventing fibrosis or a fibrosis-associateddisease.

The invention further pertains to a protein oligomer comprising (i) atleast two NC-1 monomers of collagen 18 or (ii) at least two endostatindomains of collagen 18 or (iii) at least two N-terminal peptides of thecollagen 18 endostatin domain, for use in treating, ameliorating orpreventing a vascular endothelial growth factor (VEGF)-related diseaseor a matrix metalloproteinase (MMP)-related disease.

The term “protein” or “polypeptide” or “peptide” (all terms are usedinterchangeably, if not indicated otherwise) as used herein encompassesisolated and/or purified (poly)peptides being essentially free of otherhost cell polypeptides. The term “peptide” as referred to hereincomprises at least two, three, four, five, six, seven, eight, nine, ten,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 70, 80, 90, 100,150, 200, 250, 300 or even more amino acid residues where the alphacarboxyl group of one is bound to the alpha amino group of another. Apost-translational modification of the protein or peptide as used hereinis the modification of a newly formed protein or peptide and may involvedeletion of amino acids, chemical modification of certain amino acids,for example, amidation, acetylation, phosphorylation, glycosylation,formation of pyroglutamate, oxidation/reduction of sulfa group on amethionine, or addition of similar small molecules, to certain aminoacids.

The term “protein” or “peptide” as used herein encompassespeptidomimetics. As known in the art, peptidomimetics are compoundswhose essential elements (pharmacophore) mimic a natural peptide orprotein in 3D space and which retain the ability to interact with thebiological target (such as Fibronectin) and produce the same biologicaleffect (for example, anti-fibrotic activity as defined herein); see,e.g., the review by Vagner et al. 2008, Current Opinion in ChemicalBiology 12, Pages 292-296. Peptidomimetics are designed to circumventsome of the problems associated with a natural peptide, e.g., stabilityagainst proteolysis (duration of activity) and poor bioavailability.Certain other properties, such as selectivity for the biological target,such as Fibronectin, or potency of the biological activity, such asanti-fibrotic activity, often can be substantially improved.

Protein or peptide modifications as used herein include syntheticembodiments of (poly)peptides described herein. In addition, analogs(non-peptide organic molecules), derivatives (chemically functionalized(poly)peptide molecules obtained starting with the disclosed(poly)peptide sequences) and variants (homologs) of these proteins canbe utilized in the methods and medical and diagnostic uses describedherein. Each (poly)peptide of this disclosure is comprised of a sequenceof amino acids, which may be either L- and/or D-amino acids, naturallyoccurring and otherwise. Peptides can be modified by a variety ofchemical techniques to produce derivatives having essentially the sameactivity, e.g. anti-fibrotic activity, as the unmodified (poly)peptides,and optionally having other desirable properties. For example,carboxylic acid groups of the protein, whether carboxyl-terminal or sidechain, can be provided in the form of a salt of apharmaceutically-acceptable cation or esterified to form a C₁-C₁₆ ester,or converted to an amide of formula NR₁R₂ wherein R₁ and R₂ are eachindependently H or C₁-C₁₆ alkyl, or combined to form a heterocyclicring, such as a 5- or 6-membered ring. Amino groups of the polypeptide,whether amino-terminal or side chain, can be in the form of apharmaceutically-acceptable acid addition salt, such as the HCl, HBr,acetic, benzoic, toluene sulfonic, maleic, tartaric and other organicsalts, or can be modified to C₁-C₁₆ alkyl or dialkyl amino or furtherconverted to an amide. Hydroxyl groups of the polypeptide side chainsmay be converted to C₁-C₁₆ alkoxy or to a C₁-C₁₆ ester usingwell-recognized techniques. Phenyl and phenolic rings of the polypeptideside chains may be substituted with one or more halogen atoms, such asfluorine, chlorine, bromine or iodine, or with C₁-C₁₆ alkyl, C₁-C₁₆alkoxy, carboxylic acids and esters thereof, or amides of suchcarboxylic acids. Methylene groups of the polypeptide side chains can beextended to homologous C₂-C₄ alkylenes. Thiols can be protected with anyone of a number of well-recognized protecting groups, such as acetamidegroups. Those skilled in the art will also recognize methods forintroducing cyclic structures into the (poly)peptides of this inventionto select and provide conformational constraints to the structure thatresult in enhanced stability.

The protein or (poly)peptide as referred to herein can also be a fusionprotein. The term “fusion protein” as used herein denotes a chimericprotein (literally, made of parts from different sources) which iscreated through the joining of two or more genes that originally codedfor separate proteins. Translation of this fusion gene results in asingle or multiple polypeptides with functional properties derived fromeach of the original proteins. For example, the fusion protein as usedherein comprises, e.g., a NC-1 monomer of collagen 18 and a Fc region ofan immunoglobulin, a NC-1 monomer of collagen 18 and a Fc dimer, anendostatin domain of collagen 18 and a Fc region of an immunoglobulin,an endostatin domain of collagen 18 and a Fc dimer, an endostatin domainof collagen 18 with a single mutation at position 7 in which glutamineis replaced by cysteine and a Fc region of an immunoglobulin, anendostatin domain of collagen 18 with a single mutation at position 7 inwhich glutamine is replaced by cysteine and a Fc dimer, a N-terminalpeptide of the collagen 18 endostatin domain and a Fc region of animmunoglobulin or a N-terminal peptide of the collagen 18 endostatindomain and a Fc dimer. For example, it can be advantageous to express aNC-1 monomer of collagen 18 and a Fc region of an immunoglobulintogether with a Fc region of an immunoglobulin in a cell to avoiduncontrolled aggregation of the NC-1. Or it can be appropriate to omitor mutate the native association region in NC-1 so that NC-1 cannotoligomerize any longer via this association region. If such a NC-1without functional association region is fused to a Fc region, thisresults in the formation of a NC-1 dimer. The fusion protein canadditionally encompass, e.g., a RGD motif and/or a PHSRN motif ofFibronectin. The terms “NC-1 monomer of collagen 18”, “endostatin domainof collagen 18”, “N-terminal peptide of the collagen 18 endostatindomain”, “Fc region”, “RGD motif” and “PHSRN motif of Fibronectin” aredefined elsewhere herein. Further fusion proteins encompassed by thepresent invention are indicated elsewhere in the specification and thefollowing Examples. Recombinant fusion proteins are created, e.g., byrecombinant DNA technology well described in the art see, e.g., Sambrooket al., Molecular cloning: a laboratory manual/Sambrook, Joseph;Russell, David W.—. 3rd ed.—New York: Cold Spring Harbor Laboratory,2001.

The term “oligomer” usually refers to a macromolecular complex formed bynon-covalent bonding of a few macromolecules like proteins or nucleicacids, in biochemistry. A dimer is per definition a macromolecularcomplex formed by two, usually non-covalently bound, molecules likeproteins or peptides. Such a complex can also be formed by proteindomains which are parts of protein sequences and structures that canevolve, function, and exist independently of the rest of the proteinchain(s). A homo-dimer is formed by two identical molecules. Theunderlying process is called homo-dimerization. A hetero-dimer is builtby two different molecules which are formed by hetero-dimerization. Asknown in the art, most dimers or trimers in biochemistry are notconnected by covalent bonds, with the exception of disulfide bridges.Some proteins contain specialized domains to ensure dimerization,trimerization or oligomerization, so called dimerization, trimerizationor oligomerization domains, as further defined herein below, and wellknown in the art. To provide an example, dimerization can be mediated byan Fc domain of an immunoglobulin or by disulfide bridges or, both or,other means as described elsewhere herein. For instance, Fc-endostatin(FcE) as used in Example 11 consists of two Fc chains (connected bydisulfide bonds), extended to two molecules of endostatin each linked toa single Fc chain. Therefore, the two adjacent endostatin moleculesbecome a dimer as a result of the Fc dimer. Another dimeric constructused in Example 11 comprised two endostatin domains of collagen 18. Eachendostatin domain contained a single mutation at position 7 in whichglutamine was replaced by cysteine. Each endostatin domain was linked toan Fc region of an immunoglobulin, with an intervening enterokinasecleavage site. In this construct, both Fc and endostatin molecules weredimerized by their corresponding disulfide bonds. Enterokinase digestionof this recombinant protein resulted in an Fc dimer and an endostatindimer. A trimer is a macromolecular complex formed by three, usuallynon-covalently bound peptides, proteins or protein domains. Forinstance, for trimerization, the native association region within theNC-1 domain can be used which mediates the trimerization of NC-1 ofcollagen 18 because the native association region within the NC-1 domainof collagen 18 functions as a trimerization domain. A homo-trimer isformed by three identical molecules, whereas a hetero-trimer is built bythree different molecules. For instance, collagen 18 is a homo-trimericprotein. A tetramer consists of four molecules, a pentamer of fivemolecules, and so on. In these cases, complex formation is oftenmediated by oligomerization domains, as set forth above.

In the context of the present invention, an “oligomer” is to beunderstood as a “protein oligomer” or “peptide oligomer” that comprisesa few monomer units, e.g., two, three, four, five or even more monomerunits. Accordingly, the oligomer can be, e.g., a dimer, trimer,tetramer, pentamer, and so on. Preferably, the oligomer is a homo-dimer,homo-trimer etc. The monomer unit (or briefly monomer) can be, e.g., anNC-1 monomer of human or murine collagen 18, an endostatin domain ofhuman or murine collagen 18, or an N-terminal peptide of the human ormurine collagen 18 endostatin domain, as specified elsewhere herein. Themonomer can also be an NC-1 monomer of rhesus, macaque or primatecollagen 18, an endostatin domain of rhesus, macaque or primate collagen18, or an N-terminal peptide of the rhesus, macaque or primate collagen18 endostatin domain.

The monomer can also be a fusion protein comprising an NC-1 monomer ofhuman, rhesus, macaque, primate or murine collagen 18, an endostatindomain of human, rhesus, macaque, primate or murine collagen 18, or anN-terminal peptide of the human, rhesus, macaque, primate or murinecollagen 18 endostatin domain. The term “fusion protein” as used hereincomprises at least one NC-1 monomer, at least one endostatin domain, orat least one N-terminal endostatin peptide or peptide derived from theN-terminus of the endostatin domain, as defined herein. Encompassed arealso fusion proteins comprising two, three of even more NC-1 monomers,two, three or even more endostatin domains, or two, three of even moreN-terminal endostatin peptides or peptides derived from the N-terminusof the endostatin domain, as defined herein For example, said at leastone NC-1 monomer, endostatin domain or N-terminal endostatin peptide orpeptide derived from the N-terminus of the endostatin domain can belinked to a Fc domain from an immunoglobulin, a purification tag, alabel, another therapeutic agent, such as an anti-fibrotic agent, ananti-angiogenic agent and/or anti-tumorigenic agent, or the like. A“label” as referred to herein is a detectable compound or compositionthat is conjugated directly or indirectly to another molecule, such as aNC-1 monomer of human, rhesus, macaque, primate or murine collagen 18,an endostatin domain of human, rhesus, macaque, primate or murinecollagen 18, or an N-terminal peptide of the human, rhesus, macaque,primate or murine collagen 18 endostatin domain, to facilitate detectionof that molecule. Specific, non-limiting examples of labels includefluorescent tags, enzymatic linkages, and radioactive isotopes.Preferably, the fusion protein is human, rhesus, macaque or primate,more preferably human. The Fc domain from the immunoglobulin can befused either to the N-terminus or the C-terminus of the monomer asdefined herein, preferably to the N-terminus.

The term “protein oligomer” (or “peptide oligomer”) as used hereinincludes also protein preparations comprising the protein oligomer orpeptide oligomer and other proteins, agents or compounds, in addition.For example, said oligomer as defined herein can be administered to asubject in the need thereof, in a combination regimen, using one or morefurther anti-fibrotic, anti-angiogenic and/or anti-tumorigenicprotein(s), compound(s) or agent(s). Combinations of medications areoften more effective against fibrosis or fibrosis-associated diseasesthan a single medication used alone. To provide an example, the proteinoligomer or peptide oligomer as defined herein can be used incombination with angiostatin or an angiostatin fusion protein, such asangiostatin linked to an Fc domain of an immunoglobulin, or togetherwith inhibitors of other pathways associated with the fibrosis process,including, for example, inhibitors of TGF-beta, PDGF, VEGF, mTOR, CTGF,integrins, matrix-metalloproteinases, anti-inflammatory agents such assteroids inhibitors of cyclooxygenase, IKK/NFkB. JAK/STAT, and/or Pi3Ksignaling.

The term “collagen 18” and “collagen XVIII” are used interchangeablyherein and refer to the same protein. Collagen 18 consists of a central,interrupted triple-helical domain, flanked at the N-terminus (NC-11domain) and C-terminus (NC-1 domain), by larger non-triple helical,globular structures (Oh et al., PNAS 1994, 91, 4229; Oh et al., Genomics1994, 19, 494; Abe et al. 1993, Biochem. Biophys. Res. Commun. 196,576). The Type XVIII collagen belongs to a unique and novel subclass ofthe collagen superfamily for which the name “MULTIPLEXIN family” hasbeen proposed.

The cloning of the mouse and human collagen 18 proteins has beendescribed by Oh et al. (loc. cit.). The nucleotide and amino acidsequences of mouse collagen 18 are shown in accession numberNM_001109991.1, whereas the corresponding human sequences are shown inNM_030582.3. Further, the amino acid sequences of mouse and humancollagen 18 are shown in SEQ ID NOs: 1 and 2, respectively. Thenucleotide and amino acid sequences of Macaca mulatta collagen 18 areshown in UniProt accession number I0FVB6. Preferably, collagen 18 asreferred to herein is human collagen 18.

The “NC-1 domain” (or briefly NC-1 or NC1) as used herein is derivedfrom or is from the C-terminus of collagen 18 and includes (i) anN-terminal association region (of about 50 amino acid residues), (ii) acentral protease-sensitive hinge region (of about 70 amino acidresidues) and/or (iii) a C-terminal stable endostatin domain (of about180 amino acid residues) (Sasaki et al., 1998, EMBO J. 17, 4249).

A (poly)peptide “derived from” the NC-1 domain as used herein means thatsuch a (poly)peptide is identical to or can differ from thecorresponding amino acid sequence of the native (poly)peptide in theNC-1 domain, in one, two, three, four, five, six, seven, eight, nine,ten, 15, 20, 25, 30, 35, 40, 50 or even more amino acid residues, whileat least maintaining (or even exceeding) the biological activity (asdescribed elsewhere herein) of the corresponding NC-1 domain, such asthe oligomerization properties, anti-angiogenic, anti-tumorigenic and/oranti-fibrotic activity. The mentioned term (poly)peptide “derived from”the NC-1 domain comprises variants of the NC-1 domain, as definedelsewhere herein.

The amino acid sequence of the NC-1 domain of the mouse collagen 18 isdepicted in SEQ ID NO: 3, whereas the corresponding sequence of the NC-1domain of human collagen 18 sequence is shown in SEQ ID NO: 4.

The “association domain” or “association region” (both terms areinterchangeably) of the human NC-1 domain comprising amino acid residuesfrom about 10 to about 60 of the amino acid sequence shown in SEQ ID NO:4 is responsible for non-covalent trimerization of the NC-1 monomer toform a globular trimer. Accordingly, this association domain functionsas a trimerization domain. The proteolytic cleavage-sensitive “hingeregion” comprises amino acid residues from about 61 to about 129 of theamino acid sequence shown in SEQ ID NO: 4. The compact “endostatindomain” comprises amino acid residues from about 130 to about 308 of theamino acid sequence shown in SEQ ID NO: 4; see, e.g., Sasaki, loc. cit.;Kuo 2001, JCB 152, 1233; Tjin et al. 2005, Cancer Res 65, 3656. Theendostatin domain comprises a zinc binding site which mediates bindingto zinc and is located at the N-terminus of endostatin (Ding et al.,1998, PNAS 95, 10443; U.S. Pat. No. 7,524,811). Interestingly, this zincbinding site has been shown to be responsible for theanti-tumor/anti-angiogenic activity of endostatin (Boehm et al., 1998,Biochem. Biophys. Res. Commun. 252, 190). The association region and theendostatin domain in the NC-1 domain are connected by the hinge region(see Sasaki et al., loc. cit.). The hinge region has been found to becleaved, for instance, by matrix metalloproteinases (MMPs), such asMMP-3, -7, -9, -13 and -20 (Heliasvaara et al., Exp Cell Res 2005, 307,192).

The above-indicated domain structure of NC-1 is based on structuraldata. The term “about” as used for the positioning of the domains withinNC-1 reflects the fact that the exact boundaries between the mentioneddomains may differ from the indicated positions by one, two, three,four, five or even more amino acid residues. However, the exact boundarybetween, for example, the association domain and the hinge region can bedetermined by generating an association domain comprising amino acidresidues from about 10 to about 60 of SEQ ID NO: 4 as a starting point,and producing shorter fragments thereof, e.g., with a length of 49, 48,47, 46, 45 and so on, amino acid residues. Said shorter fragments canthen be analyzed for their oligomerization properties, i.e. whether theyare still able to form oligomers, such as trimers, as the completeassociation domain does.

Alternatively, the endostatin domain may serve as a starting point toaddress the oligomerization properties of the domains of NC-1. Forexample, a method for identifying the exact boundaries of the monomer,dimer and/or trimer transitions in the NC-1 domain as defined herein,can comprise: a) generating a series of recombinant peptides from orderived from the NC-1 domain, starting with a peptide consisting of theendostatin domain, followed by increasing the size of said peptideconsisting of the endostatin domain in steps of about 10 to 20 aminoacid residues, and b) testing the recombinant peptides of step a) fortheir oligomerization properties, i.e. whether said peptides are able toform dimers or trimers and identifying peptides which are able to formsuch oligomers, and c) determining the exact boundaries of the monomer,dimer and/or trimer transitions in the NC-1 domain. The method couldcomprise a further step d) of constructing an oligomer using therecombinant peptides identified in step b) which are able to form dimersor trimers. For generating a series of recombinant peptides from orderived from the NC-1 domain, peptide or protein synthesis known in theart can be used. For testing the oligomerization properties of proteinoligomers or peptide oligomers as defined herein, for example, Westernblot analysis, immunoprecipitation, SDS-PAGE, chromatographic methods orother methods well known in the art can be utilized; see, e.g., Sambrooket al., Molecular cloning: a laboratory manual/Sambrook, Joseph;Russell, David W.—. 3rd ed.—New York: Cold Spring Harbor Laboratory,2001. Accordingly, the person skilled in the art is able to identify theexact boundaries of the monomer, dimer and/or trimer transitions in theNC-1 domain as defined herein, without undue burden. In addition, theabove-mentioned domain model fits the gene structure remarkably well,with exons 38 and 39 encoding the association domain, exon 40 the hingeregion, and three more exons the endostatin domain (Sasaki et al., loc.cit.).

The recombinant (poly)peptides generated by the above-indicated methodscan be used to produce oligomers or fusion proteins, such as Fc fusionproteins, which form such oligomers and which can then further be testedfor their anti-fibrotic activity, anti-angiogenic activity,anti-invasive/anti-metastatic activity, reducing vascular permeabilityactivity, anti-inflammatory and/or anti-tumorigenic activity . Anoligomer comprising such recombinant (poly)peptides or fusion proteinsis particularly useful as a pharmaceutical composition for treating,ameliorating or preventing fibrosis or fibrosis-associated diseases,vascular endothelial growth factor (VEGF)-related diseases or matrixmetalloproteinase (MMP)-related diseases, as defined elsewhere herein.

The term “NC-1 monomer of collagen 18” as referred to herein, comprisesan oligomerization domain, a hinge region and/or an endostatin domain.

The term “oligomerization domain” as used herein refers generally to aprotein domain which mediates the sub-unit assembly of the two or moreNC-1 monomers, as defined herein. As indicated above, theoligomerization domain mediates dimerization, trimerization, ortetramerization and so on, of the NC-1 monomers. Such oligomerizationleads, e.g., to functional advantages of multivalency and high bindingstrength, increased structure stabilization and combined functions ofdifferent domains, resulting in enhanced biological activity, such asimproved or increased anti-fibrotic activity, anti-angiogenic activity,anti-invasive/anti-metastatic activity, reducing vascular permeabilityactivity, anti-inflammatory and/or anti-tumorigenic activity, incomparison to the NC-1 monomer. The oligomerization domain can comprise,e.g., the association domain of the NC-1 domain mentioned above, i.e.the non-triple helical trimerization domain of collagen 18 which isresponsible for non-covalent oligomerization of the NC-1 monomers or thecollagen 18 helices. To give an example, the association domain of thehuman NC-1 domain can comprise amino acid residues from about 10 toabout 60, preferably from amino acid residues 10 to 60, of the aminoacid sequence shown in SEQ ID NO: 4, or peptides thereof. Theassociation domain or peptides thereof are capable of mediating thenon-covalent trimerization of the NC-1 monomer. The trimerizationproperties of the association domain or peptides thereof can be testedby methods mentioned elsewhere herein which are also known in the art(Sambrook, loc. cit.). In further aspects, the oligomerization domaincan comprise other scaffold constructs/domains providing oligomerizationand longer half life, well described in the literature; see, e.g. thereview by Ali and Imperiali 2005, Bioorganic and Medicinal Chemistry 13,5013. Such an oligomerization domain replaces structurally andfunctionally the association domain as found in the natural human NC-1domain referred to above, or is used, in addition, to said associationdomain, in the protein or peptide oligomer. The oligomerization canalternatively be mediated by an Fc domain of an immunoglobulin, i.e. theoligomerization domain of the NC-1 monomer as defined herein comprisesor is an Fc domain of an immunoglobulin. It is known in the art thatfusion of an Fc domain to, e.g., a peptide or protein mediates a longerhalf life in circulation. It is to be understood that the Fc domain maybe used in said NC-1 monomer, in addition to the association domain ofthe NC-1 domain mentioned above or may replace the association domain.The Fc domain confers a dimeric structure on the NC-1 monomer as definedherein since Fc is a dimer itself. In another aspect, theoligomerization can be mediated by the introduction of a structuralmodification, e.g., a mutation into the NC-1 monomer which results inthe formation of disulfide bonds, as set forth in more detail below. Itis further envisaged that the protein oligomers or peptide oligomers canbe formed covalently.

The “hinge region” as referred to herein comprises amino acid residuesfrom about 61 to about 129, preferably from amino acid residue 61 to129, of the amino acid sequence shown in SEQ ID NO: 4, or peptidesthereof. The hinge region or peptides thereof can comprise at least oneendogenous proteolytic cleavage site such as an MMP-3, -7, -9, -13 or-20 cleavage site. Optionally, the hinge region within the NC-1 monomerof collagen 18 can comprise one or more recombinant protease cleavagesites, in addition to the endogenous protease cleavage sites of thehinge region. Such a recombinant protease cleavage site can be, forinstance, an enterokinase, factor Xa or thrombin cleavage (Bergers andJavaherian; Lee et al.; loc. cit.). For example, cleavage by therespective protease allows for the release of the endostatin domain orfragments thereof such as (an) N-terminal endostatin peptide(s) from theprotein oligomer or peptide oligomer. The hinge region or peptidesthereof can comprise also more than one proteolytic cleavage site, suchas two, three, four or even more proteolytic cleavage sites.

The hinge region can be interposed between the oligomerization domainand the endostatin domain or fragment(s) of said endostatin domain suchas N-terminal endostatin peptides. Preferably, the hinge region islocated between the oligomerization domain and the endostatin domain orfragment(s) thereof, in the NC-1 monomer as referred to herein. Thedomain arrangement within the NC-1 monomer of collagen 18 can beoligomerization domain-hinge region-endostatin domain or fragment(s) ofsaid endostatin domain, or endostatin domain or fragment(s) of saidendostatin domain-hinge region-oligomerization domain.

The mentioned NC-1 monomer can also comprise the oligomerization domainand an endostatin domain. In this case, the hinge region is missing.

The “endostatin domain of collagen 18” as used herein comprises aminoacid residues from about 130 to about 308, preferably from 130 to 308,of the amino acid sequence shown in SEQ ID NO: 4. The correspondingamino acid sequence of said endostatin domain can be derived, forinstance, from UniProtKB accession number P39060 (human) and P39061(murine). Moreover, the endostatin domain of collagen 18 as used hereincomprises SEQ ID NO: 18 in FIG. 2 which shows the amino acid sequence ofmurine endostatin; and SEQ ID NO: 19 in FIG. 2 which shows the aminoacid sequence of human endostatin. Encompassed by the scope of theinvention is also an endostatin domain of collagen 18 in which theglutamine at position 7 of SEQ ID NO: 18 or 19 is replaced by cysteine,resulting in an endostatin dimer covalently attached by a disulfidebond. If not otherwise specified, the term “endostatin” used hereinrefers to the endostatin domain of collagen 18.

The NC-1 monomer can also comprise a fragment of said endostatin domain,instead of the complete endostatin domain. For example, the fragment canbe an N-terminal peptide of the endostatin domain of collagen 18, asdefined herein. The NC-1 monomer comprises at least one N-terminalpeptide of the endostatin domain of collagen 18 but can also comprisetwo, three, four or even more N-terminal endostatin peptides. TheN-terminal endostatin peptides can be identical or different.Encompassed are linear, branched or cycled N-terminal endostatinpeptides. For example, it is envisaged that two, three or moreN-terminal endostatin peptides can be arranged in linear form. Theendostatin domain or N-terminal peptide thereof has (at least)anti-fibrotic activity. In addition, the endostatin domain or N-terminalpeptide thereof can have anti-angiogenic activity,anti-invasive/anti-metastatic activity, reducing vascular permeabilityactivity, anti-inflammatory and/or anti-tumorigenic activity.

The term “N-terminal peptide of the collagen 18 endostatin domain” asused herein means a peptide from or derived from the amino (N orNH₂)-terminus of the endostatin domain of collagen 18. The N-terminus ofthe endostatin domain of collagen 18 comprises amino acid residues fromabout amino acid residue 1 to about amino acid residue 132, preferablyfrom amino acid residue 1 to amino acid residue 132, of SEQ ID NO: 18(corresponding to the murine endostatin domain of collagen 18) orpreferably SEQ ID NO: 19 (corresponding to the human endostatin domainof collagen 18). Further encompassed are shorter fragments thereof, i.e.at least two, three, four, five, six, seven, eight, nine, ten, 11, 12,13, 14, 15, 16, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 46, 47, 48, 49, 50,60, 70, 80, 90, 100, 110, 120 or 130 amino acid residues. For example,polypeptide comprising amino acid residue 1 to amino acid residue 132,of SEQ ID NO: 19 (corresponding to the human endostatin domain ofcollagen 18) may serve as a starting point to address the anti-fibroticactivity, anti-angiogenic activity, anti-invasive/anti-metastaticactivity, reducing vascular permeability activity, anti-inflammatoryand/or anti-tumorigenic activity of such shorter fragments.Subsequently, a series of recombinant peptides from or derived from thehuman endostatin domain is generated, starting with a peptide consistingof the human endostatin domain, followed by reducing the size of saidpeptide consisting of the human endostatin domain in steps of about 5 to20 amino acid residues, and analyzing these recombinant peptides for thementioned activities. Tests for analyzing said biological activities aredescribed elsewhere herein and known in the art. Preferably, theN-terminal peptide of the collagen 18 endostatin domain comprises orconsists of the amino acid sequence shown in SEQ ID NO: 20 (murine) orSEQ ID NO: 22 (human); see also FIG. 2 . In one aspect, the N-terminalpeptide of the collagen 18 endostatin domain comprises an N-terminalendostatin peptide without change of the amino acid sequence, incomparison to the wild-type N-terminal endostatin peptide. Put itdifferently: The amino acid sequence of such peptide can be found in thenative endostatin domain of NC-1. A “peptide derived from the N-terminusof the endostatin domain of collagen 18” comprises a peptide or peptidevariant which can differ from the corresponding native endostatinpeptide in the endostatin domain of NC-1, in one, two, three, four, fiveor even more amino acid residues, while at least maintaining (or evenexceeding) the biological activity (e.g., anti-fibrotic activity,anti-angiogenic activity, anti-invasive/anti-metastatic activity,reducing vascular permeability activity, anti-inflammatory and/oranti-tumorigenic activity) of the corresponding endostatin peptide, inthe endostatin domain of NC-1. Such a peptide is also referred to hereinas an “N-terminal endostatin-derived peptide” or “variant of aN-terminal peptide of the collagen 18 endostatin domain” and isencompassed by the term “N-terminal peptide of the collagen 18endostatin domain” as used herein. The peptide from or derived from theamino-terminus of the endostatin domain of collagen 18 can exhibitfurther modifications, as described elsewhere herein. For example, itcan be fused to an Fc domain of an immunoglobulin or can contain anotheroligomerization domain as defined herein in order to mediatedimerization or oligomerization of the fusion protein. The fusionprotein can include further therapeutic agents as mentioned elsewhere inthis specification.

The term “treatment” as used herein denotes the improvement or evenelimination of one or more symptoms associated with the disease asreferred to herein, by the administration of a protein oligomer orpeptide oligomer or fusion protein as defined herein to a subject in theneed thereof.

The term “amelioration” as referred to herein means the act of makingbetter or improving the disease as referred to herein in the subject, byadministering the protein oligomer or peptide oligomer or fusion proteinas specified herein. An improvement may also be seen as a slowing orstopping of the progression of the disease.

The term “prevention” as utilized herein means the avoidance of theoccurrence or re-occurrence of the disease referred to herein, by theadministration of a protein oligomer or peptide oligomer or fusionprotein as defined herein.

The term “fibrosis-associated disease” as used herein denotes anydisorder associated with fibrosis. The fibrosis-associated disease ispreferably selected from the group consisting of: fibrosis of the skin,preferably scleroderma; keloid or keloid scar; hypertrophic scar;morphea; fibrosis as a result of graft-versus-host disease;subepithelial fibrosis; endomyocardial fibrosis; uterine fibrosis;myelofibrosis; retroperitoneal fibrosis; nephrogenic systemic fibrosis;scarring after surgery; asthma; cirrhosis/liver fibrosis; fibrosis as aresult of aberrant wound healing; glomerulonephritis; endometriosis,multifocal fibrosclerosis; radiation-induced fibrosis (as an example forstimulation-induced fibrosis), preferably radiation-induced pneumonitisor radiation-induced lung fibrosis; chemotherapy-induced or drug-inducedfibrosis, e.g., as a result of mTOR or EGFR kinase inhibition; usual oridiopathic pulmonary fibrosis (as an example for idiopathic fibrosis);fibrosis as the result of autoimmune diseases, e.g., Lupus,intra-tumoral- and cancer-associated fibrosis/fibrogenesis, organfibrosis-followed chronic inflammation (e.g., via viral stimulus ortransplantation); organ fibrosis as the endstage of chronic kidneydiseases, long term dialysis, or diabetes mellitus (PMID:15939343,Common pathways in idiopathic pulmonary fibrosis and cancer, Eur RespirRev 2013 22:265-272, Nat Rev Nephrol. 2014 Mar 25. doi:10.1038/nrneph.2014.31. PMID:24662433 and PMID:23938596, Nat RevNephrol. 2014 Apr;10(4):226-37. doi: 10.1038/nrneph.2014.14. Epub 2014Feb 11. PMID:24514753, Nat Rev Gastroenterol Hepatol. 2014 Jan;11(1):4.doi: 10.1038/nrgastro.2013.227. Epub 2013 Nov 26. PMID:24275791).

Idiopathic pulmonary fibrosis (IPF) is a condition also known ascryptogenic fibrosing alveolitis (CFA) that is a chronic, progressiveform of lung disease characterized by fibrosis of the supportingframework (interstitium) of the lungs. By definition, the term is usedonly when the cause of the pulmonary fibrosis is unknown (“idiopathic”).When lung tissue from patients with IPF is examined under a microscopeby a pathologist, it shows a characteristic set of histologic/pathologicfeatures known as usual interstitial pneumonia (UIP). UIP ischaracterized by progressive scarring of both lungs that involves thesupporting framework (interstitium) of the lung.

The protein oligomer or peptide oligomer or fusion protein as referredto herein has at least anti-fibrotic activity. An “anti-fibroticactivity” as used herein means a biological activity which causesslowing down, stopping or even regression of fibrosis or afibrosis-associated disease. Preferably, the fibrosis orfibrosis-associated disease is cured by the anti-fibrotic activity ofthe protein or peptide oligomer or fusion protein as defined herein.“Fibrosis” is the formation or development of excess fibrous connectivetissue in an organ or tissue as a reparative or reactive process (e.g.,in reaction to radiation and/or chemotherapy), contrary to a formationof fibrous tissue as a normal constituent of an organ or tissue. Forexample, treatment-related reductions in fibrosis can be associated withmodulation of cytokines and growth factors.

The anti-fibrotic activity of a protein oligomer or peptide oligomer orfusion protein as defined herein can be tested, e.g., by animal modelsknown in the art and shown in the following examples. For instance,bleomycin- and radiation-induced lung fibrosis models have been used tostudy lung fibrosis (Rubin, loc. cit.; Kamp, loc. cit. Gurujeyalakshmiet al. (1996), Res. Commun. Pharmacol. Toxicol. 1, p. 1-15;Gurujeyalakshmi et al. (1999), Am. J. Physiol. 276, p. L311-L318;Hallahan et al. (2002), J. Natl. Cancer Inst. 94, p. 733-741).Anti-fibrotic activity of a protein oligomer or peptide oligomer orfusion protein as specified herein can also be analyzed histologically(reduction or diminishing inflammation, granuloma formation and/orfibrosis), by analysis of expression of growth factors and/or cytokines(e.g., inhibition of PDGF-receptor activation, reduction of transforminggrowth factor-β, inhibition of receptor tyrosine kinase activation,reduction of IL-1β, KC, or TIMP-1), reduction of collagen, inhibition offibroblast proliferation and fibroblast to myofibroblast transformation,and/or reduction of BAL lymphocytes, reduction of qualitative andquantitative surrogates of fibrosis (e.g. increase of lung density[Hounsfield units] and decrease of lung volume) using high resolutioncomputer tomography, improved oxygen saturation (analysis ofblood-gases, pulse-oximetry), Pulmonary Function Testing (PFT) andclinical symptoms (breath rate, heart rate, signs of right heartfailure, diffusion capacity, Spirometry).

The term “vascular endothelial growth factor (VEGF)-related disease” asused herein denotes benign pathophysiological conditions depending onderegulation of the VEGF levels such as wet macular degeneration,endometriosis, bronchial asthma and diabetes mellitus, enhanced VEGFinduced vascular permeability (e.g. enhanced permeability afterirradiation of brain tissue, “radionecrosis”), alterations of vaso-tonus(e.g. hypertension), rheumatoid arthritis etc., as well as malignantVEGF dependent diseases such as renal cell cancer and other VEGFaddicted tumors, VEGF dependent development of ascites, VEGF dependentsuppression of immune system, e.g. recruitment and microenvironmentaleducation of bone marrow-derived cells (BMDC), myeloid derivedsuppressor cells (MdSC), immature dendritic cells, etc. Preferably, thevascular endothelial growth factor (VEGF)-related disease is aVEGF-A-related disease.

The term “matrix metalloproteinase (MMP)-related disease” as referred toherein means benign and malignant diseases where MMP activationcontributes to the pathophysiology, e.g., activation of MMPs during theprocess of local tumor invasion and cancer metastasis inherently evidentin tumors with high local therapy failure rates such as glioblastoma,pancreatic cancer, lung cancer, as well as acquired enhanced MMPactivation as the function of therapy induced selection pressures (e.g.tumor hypoxia and fibrosis post radiotherapy), overt immune reaction inautoimmune diseases and chronic inflammatory diseases etc. Preferably,the matrix metalloproteinase (MMP)-related disease is aMMP-2/MMP-9-related disease.

In a preferred embodiment, the protein oligomer or peptide oligomer orfusion protein as referred to herein has one, two or even morebiological activities, in addition to the anti-fibrotic activity,wherein the additional biological activity is selected from the groupconsisting of: anti-angiogenic activity, anti-invasive/anti-metastaticactivity, reducing vascular permeability activity, anti-inflammatory andanti-tumorigenic activity.

In addition to the anti-fibrotic activity, the protein oligomer orpeptide oligomer or fusion protein as defined herein can haveanti-angiogenic activity, anti-invasive/anti-metastatic activity,reducing vascular permeability activity, anti-inflammatory and/oranti-tumorigenic activity. Such activities include, for example, anybiological activity inhibiting the growth or migration of endothelialcells and/or pericytes, formation of tubes or endothelium, growth of newcapillary blood vessels in the body, slowing or inhibiting of the growthof benign or malignant tumors by cutting off their blood supply, reduceside-effects/toxicity of other anti-tumor or anti-angiogenic agents,e.g., VEGF-inhibitors, by interference with their mechanism of action,i.e. reduce blood pressure, modulation of inflammatory response inmalignant and benign diseases, or improving the pathophysiologicalparameters, such as perfusion or hypoxia within a therapeutic timewindow after treatment that, in turn, may facilitate the efficacy ofadditional therapies (e.g., radiotherapy, chemotherapy or anti-apoptotictherapy). The anti-angiogenic activity can be tested by in vitro assaysor in vivo by animal models known in the art (Abdollahi et al., CancerRes. 2003, 63, 8890; Mol. Cell 2004, 13, 649; PNAS 2007, 104, 12890;Drug Resist. Update 2005, 8, 59; Bergers et al., Science 1999, 284, 808;Javaherian et al., J. Biol. Chem. 2002, 277, 45211; Lee et al., Clin.Cancer Res. 2008). For instance, the anti-angiogenic activity can betested in vitro by inhibition of the proliferation and/or migration ofendothelial cells stimulated by a growth factor, e.g., by VEGF. In vivoanti-angiogenic activity can be analyzed, for example, by a chickenchorioallantoic membrane (CAM) assay, whereas an anti-tumor activity canbe tested in animal tumor models including, e.g., A549, LLC or H460non-small cell lung carcinoma, HT29 colon carcinoma, BxPC3 PancreaticCarcinoma, Karpas 299 lymphoma, MOLM-13 AML (acute myeloid leukemia),786-O, A2058 cell line (melanoma) or RENCA renal cell carcinoma (RCC)and many others (Abdollahi et al., Drug Resist. Update 2005, loc. cit.).

Medicaments for the therapy of a vascular endothelial growth factor(VEGF)-related disease which can be used in addition to the proteinoligomer or peptide oligomer of fusion protein of the invention include,for example, other modulators of vascular permeability (e g enhancedpermeability after irradiation of brain tissue, “radionecrosis”) andvaso-tonus (e.g. endothelin antagonists macitentan, AT1/ACE inhibitors),β2-sympathomimetics and corticoids in asthma, immune-suppressants inchronic inflammatory/auto-immune diseases, chemotherapy and radiotherapyfor different VEGF-dependent tumors and ascites, kinase inhibitors used,e.g. in renal cell cancer (mTORi e.g., RAD001, multikinase inhibitorspazopanib/suitinib/axitinib, immune modulators, e.g. checkpointinhibitors anti-PD-1/PD-L1 antibodies).

Medicaments for the therapy of a matrix metalloproteinase (MMP)-relateddisease which can be used in addition to the protein oligomer or peptideoligomer or fusion protein of the invention include, for example,locally invasive tumors with high loco-regional therapy failure ratestreated with radio-(chemo)-therapy such as glioblastoma, pancreaticcancer, anti-inflammatory and immunosuppressive therapy (anti-TNF alphaantibodies/infliximab, mycophenolic acid, cyclophosphamide etc.), tumorinvasion or pseudoprogression after cancer treatment, e.g.anti-angiogenic therapy in recurrent glioma, treatments of metastaticdiseases with high MMP-2/-9 activity such as breast cancer (i.e.hormonal therapy tamoxifen, Trastuzumab in HER2+ disease,chemotherapies).

The term “subject” as referred to in the present application pertains toa farm animal, a pet, a Macaque (such as Macaca mulatta) or a human.Preferably, the farm animal or pet is a mammal. More preferably, thesubject is a human suffering from fibrosis or a fibrosis-associateddisease, a vascular endothelial growth factor (VEGF)-related disease, ora matrix metalloproteinase (MMP)-related disease as defined herein, and,thus, is in the need for the treatment of the mentioned disease.

As used herein, the term “about” when qualifying a value of a stateditem, number, percentage, or term refers to a range of plus or minus 10percent, 9 percent, 8 percent, 7 percent, 6 percent, 5 percent, 4percent, 3 percent, 2 percent or 1 percent of the value of the stateditem, number, percentage, or term. As regards amino acid sequences, theterm “about” means plus or minus 5 amino acid residues, 4 amino acidresidues, 3 amino acid residues, 2 amino acid residues or 1 amino acidresidue. Preferred is a range of plus or minus 10 percent; or plus orminus 3 or 1 amino acid residue(s).

The terms “comprising”, “comprises” and “comprised of” as used hereinare synonyms with “including”, “includes” or “containing”, “contains”,and are inclusive or open-ended and do not exclude additional,non-recited members, elements or method steps. Evidently, the term“comprising” encompasses the term “consisting of”. More specifically,the term “comprise” as used herein means that the claim encompasses allthe listed elements or method steps, but may also include additional,unnamed elements or method steps. For example, a method comprising stepsa), b) and c) encompasses, in its narrowest sense, a method whichconsists of steps a), b) and c). The phrase “consisting of” means thatthe composition (or device, or method) has the recited elements (orsteps) and no more. In contrast, the term “comprises” can encompass alsoa method including further steps, e.g., steps d) and e), in addition tosteps a), b) and c).

The term “at least one” means one, two, three, four, five or even more.

It has been found by the present inventors in a previous study thattrimeric NC-1 (with NC-1 comprising the association domain, the hingeregion and the endostatin domain) derived from human collagen 18 bindsFibronectin, whereas endostatin monomer lacks binding to Fibronectin;see WO 2013/026913. Fibronectin is recognized as a major extracellularmatrix protein, binding angiogenic and anti-angiogenic reagents.Endostatin is a monomer under physiological conditions. The majorprecursor to endostatin is NC-1, a trimeric molecule consisting of threeinterlinked chains, each with approximately 330 amino acids. This showsthat NC-1 trimer has distinct properties, in comparison to endostatin.Furthermore, an Fc-endostatin which forms dimers as well as anartificial endostatin dimer bearing a single mutation in amino acidposition 7 (glutamine to cysteine) of endostatin retains binding toFibronectin indicating the importance of oligomerization for binding toFibronectin. Following a search for endostatin-size molecules in humansera, the inventors failed to identify the conventional size endostatin(of about 20 kDa). The appearance of endostatin size molecules in humanblood circulation might be due to the degradation of NC-1 trimer byproteases following collection of human sera. NC-1 trimer appeared to bethe major physiological product of collagen 18 degradation, present intissues and circulation showing distinct biological properties notshared by (monomeric) endostatin. The inventors further demonstratedhigh affinity binding of Fibronectin to VEGF, NC-1 trimer as well asco-immunoprecipitated these three candidate interaction partners fromperipheral blood platelets protein lysates. Furthermore, in-vivoco-localization of NC-1 trimer, Fibronectin, VEGF and alpha 5 beta 1(α5β1) integrin could be demonstrated, suggesting a model in which anensemble of VEGF, NC-1 trimer, integrin α5β1 with Fibronectin preludethe initiation of the anti-angiogenic process. Most importantly,anti-tumor studies of NC-1 trimer versus endostatin showed that NC-1trimer is a more potent anti-angiogenic protein than endostatin.

Unexpectedly, the inventors have found in a recent study, that fibrosiscan be successfully treated by a protein oligomer exemplified by anFc-endostatin fusion protein comprising an N-terminal endostatinpeptide, as shown in the following examples. This result was surprising,in light of the teaching of WO 2011/050311 (loc. cit.) and thecorresponding scientific publication by Yamaguchi et al. (Sci. Transl.Med. 2012, 4, p. 136ra71) in which anti-fibrotic activity has beenreported only for C-terminal endostatin peptides from amino acidresidues 133 to 180 (see E4 peptide). In contrast, no such activitycould be shown for N-terminal endostatin peptides.

The finding by the present inventors that the N-terminal zinc bindingregion of endostatin known to be chiefly involved in its anti-angiogeniceffects [Tjin, R. M., et al., A 27-amino-acid synthetic peptidecorresponding to the NH2-terminal zinc-binding domain of endostatin isresponsible for its antitumor activity. Cancer Research, 2005. 65(9): p.3656-3663] is also relevant for the anti-fibrotic effect elicited bythis endogenous protein is in clear contrast to recently published databy Yamaguchi et al. (loc. cit.) postulating an anti-fibrotic effect ofthe C-terminal domain of endostatin. In the radiation induced lungfibrosis (RILF) model used by the present inventors, the C-terminalpeptide was not effective to improve most investigated parameter offibrosis development. Together, the data of the present inventorsindicate an important role for the N-terminus sequence as well asdimerization of endostatin underlying its anti-fibrotic effect in theRILF model.

A closer look at the endostatin C-terminus, the E4 peptide containingarea, shows no obvious structural feature linking this fragment withpotential protein interaction partners that could provide a mechanisticexplanation for the postulated anti-fibrotic effect of the molecule.Another explanation for the lack of E4 peptide activity might be that incontrast to the acute murine fibrosis models used by Yamaguchi et al.,the present inventors utilized a radiation induced lung fibrosis model,where fibrosis development follows a slow (over 24 weeks afterirradiation) and chronic kinetic more closely resembling thepathophysiology in humans.

In the further course of the study, the most efficient attenuation oflung fibrosis, however, was found by the present inventors when asynthetic endostatin dimer (Fc-endostatin) was utilized. Fc-endostatin(FcE) consists of two Fc chains (connected by disulfide bonds), extendedto two molecules of endostatin each linked to a single Fc chain.Therefore, the two adjacent endostatin molecules become a dimer as aresult of the Fc dimer.

Based on the data shown in the following Examples, the hypothesis by thepresent inventors is that the beneficial anti-fibrotic effect of theoligomeric endostatin is at least in part mediated by its property tobind Fibronectin, and this distinguishes the NC-1 oligomers orendostatin oligomers or oligomers comprising at least two N-terminalpeptides of endostatin from monomers or monomeric fragments of NC-1 orendostatin.

In the present inventors' view, endostatin is an end-degradation productof NC-1. They present in the following Examples new data furtherdemonstrating that the binding properties of endostatin dimer and NC1trimer are quite distinct from endostatin monomer in terms of relevantprotein interaction partners.

For example, it has been found that only oligomeric endostatin and NC1is able to bind fibronectin, VEGF, MMP-2 and MMP-9, but not monomericendostatin; see also FIGS. 9 to 12 .

In other words, it is the oligomerization properties of endostatin,endostatin peptides and NC1 which plays the key role in their binding tokey players of tissue remodeling with high impact for exploration of itsanti-fibrotic and anti-cancer effects.

It will be acknowledged by those skilled in the art that this importantfinding opens a new avenue for pursuing biological properties ofoligomeric endostatin, oligomeric endostatin peptides orendostatin-derived peptides, either by synthetic design, e.g.,dimerization via Fc or other alternatives to generate and improve a drugmimicking the endostatin precursor molecule NC1 .

In one embodiment of the protein oligomer or peptide oligomer, the “NC-1monomer” or “NC-1 monomer of collagen 18” as used herein compriseseither the complete or at least one part of the non-collagenous NC-1domain of collagen 18. Preferably, the NC-1 monomer is human.

As set forth elsewhere herein, the complete C-terminal NC-1 domain ofcollagen 18 includes an N-terminal association region, a centralprotease-sensitive hinge region and a C-terminal stable endostatindomain (Sasaki et al., 1998, EMBO J. 17, 4249). The at least one part ofsaid NC-1 domain comprises at least one domain, region or fragment, ofthe non-collagenous NC-1 domain of collagen 18, preferably humancollagen 18 as depicted in SEQ ID NO: 2. Preferably, the human NC-1domain comprises or consists of SEQ ID NO: 4.

The NC-1 monomer as used herein comprises, in one aspect of the proteinoligomer or peptide oligomer, at least one N-terminal peptide of thecollagen 18 endostatin domain (briefly N-terminal endostatin peptide orN-terminal peptide) or at least one N-terminal endostatin-derivedpeptide of the collagen 18 endostatin domain.

Protein oligomer or peptide oligomer, collagen 18, the endostatin domainof collagen 18, N-terminal peptide of the collagen 18 endostatin domain,and N-terminal endostatin-derived peptide have already been definedelsewhere herein. Preferably, said oligomer, collagen 18, endostatindomain, N-terminal peptide or N-terminal endostatin-derived peptide ishuman.

Peptidomimetic and organomimetic embodiments are also envisioned,whereby the three-dimensional arrangement of the chemical constituentsof such peptido- and organomimetics mimic the three-dimensionalarrangement of the polypeptide backbone and component amino acid sidechains, resulting in such peptido- and organomimetics of the endostatindomain, N-terminal endostatin peptide or N-terminal endostatin-derivedpeptide having measurable or enhanced anti-fibrotic activity. Forcomputer modeling applications, a pharmacophore is an idealizedthree-dimensional definition of the structural requirements forbiological activity. Peptido- and organomimetics can be designed to fiteach pharmacophore with current computer modeling software (usingcomputer assisted drug design or CADD). See Walters, “Computer-AssistedModeling of Drugs,” in Klegerman & Groves, eds., 1993, PharmaceuticalBiotechnology, Interpharm Press: Buffalo Grove, Ill., pp. 165-174 andPrinciples of Pharmacology, Munson (ed.) 1995, Ch. 102, for descriptionsof techniques used in CADD. Also included are mimetics prepared usingsuch techniques.

In one aspect, the N-terminal peptide or N-terminal endostatin-derivedpeptide comprises the complete zinc binding site/domain of theendostatin domain, i.e. comprises histidines 1, 3 and 11 and asparticacid 76, of SEQ ID NO: 19. Preferably, the mentioned peptide comprisesat least histidines 1, 3 and 11 of SEQ ID NO: 19.

In another aspect, the N-terminal peptide or N-terminalendostatin-derived peptide comprises the 27 amino acid-peptide describedin Tjin, loc.cit., or fragments thereof. Preferably, the fragments ofsaid 27 amino acid-peptide comprise at least histidines 1, 3 and 11.

Further examples of N-terminal peptides or N-terminal endostatin-derivedpeptides which can be used in the protein oligomer or peptide oligomeras used herein are shown in FIG. 2 and the following examples andcomprise SEQ ID NO: 7, 9, 10, 13, 15, 18, 19, 20, or 22, or have beendescribed in the literature, e.g., in Tjin et al., loc. cit., or in EP1107989 B1 or U.S. Pat. No. 7,524,811.

Preferably, the endostatin-derived peptide or endostatin peptide isabout 10 to about 40 amino acid residues in length, preferably 15 to 35,preferably 20 to 32, more preferably 24, 25, 26, 27, 28, 29 or 30 aminoacid residues. For example, SEQ ID NO: 9 shows the corresponding murinesequence of the active motif of NC-1-endostatin domain (ED) (i.e., theamino-terminal zinc binding domain mediating anti-angiogenic and/oranti-tumor activity) with a length of 26 amino acid residues, whereasSEQ ID NO: 10 shows the corresponding human sequence with a length of 25amino acid residues. The Histidine residues in these sequences areparticularly important since it has been found by the present inventorsin a previous study, that substitution of said Histidine residues byAlanine residues abolished anti-tumor and anti-angiogenic activity.

In preferred embodiments of the protein oligomer or peptide oligomer,the NC-1 monomer comprises or consists of the endostatin domain, asdefined elsewhere herein. Preferably, the mentioned endostatin-derivedpeptide, endostatin peptide or endostatin domain comprises a singlemutation of glutamine to cysteine at position 7 of the endostatindomain. Such mutants are able to form disulfide bridges and are, thus,able to form dimers; see, e.g., Kuo 2001, JCB 152, 1233; Tjin et al.2005, Cancer Res 65, 3656. Accordingly, said mutation can be used in theprotein or peptide oligomer described herein as a means fordimerization.

In a further embodiment, the NC-1 monomer comprises a hinge region, inaddition to the endostatin domain, the endostatin-derived peptide, orthe endostatin peptide. Such a construct will probably form a monomer,possibly a dimer. The formation of a dimer cannot be excluded since itappears that the hinge region may also contribute to the dimerassociation of such constructs. Optionally, such an NC-1 monomercomprises, in addition to the mentioned constituents, an associationdomain, i.e. the non-triple helical trimerization domain of humancollagen 18, or another oligomerization domain as referred to herein. Itis evident to those skilled in the art that the presence of thementioned association domain results in the formation of a trimer. Inanother aspect, the NC-1 monomer comprises an endostatin domain and anassociation domain of the above-defined NC-1 domain and, in a stillfurther aspect, an association domain, a hinge region and an endostatindomain, each of said NC-1 domain. In the latter aspect, the NC-1 monomercomprises the complete NC-1 domain of human collagen 18 or is, i.e.consists of, the NC-1 domain of human collagen 18 (of about 38 kDa). TheNC-1 domain of human collagen 18 and the structure of said NC-1 domainhas been defined, e.g., by Sasaki et al. (loc. cit.). The NC-1 domain ofcollagen 18 consists of a non-triple-helical sequence of 315 (mouse) or312 (human) amino acid residues. As set forth above, the NC-1 domain hasbeen found to associate non-covalently to form a trimer, via theabove-mentioned association domain.

It is preferred that the NC-1 monomer as used herein is human.

Oligomerization of NC-1 is mediated by at least two domains of thisprotein: one consisting of approximately 50 amino acids at theN-terminal of the protein defining a triple-helix structure, i.e. theassociation domain. The second domain which participates inoligomerization is located at the N-terminus of endostatin and is ableto bind to zinc. The human endostatin zinc site is formed by histidines1, 3 and 11 and aspartic acid 76 of SEQ ID NO: 19. Said domain has beenshown to form a dimer at high concentration of endostatin (Ding et al.,loc. cit.). Thus, in certain aspects, the NC-1 monomer comprises theassociation domain and the N-terminus of the endostatin domain. It isalso possible that the protease sensitive hinge region plays a role inoligomerization of NC-1, as already indicated above. Accordingly, insome aspects of the invention, the NC-1 monomer can further comprise ahinge region of the NC-1 domain.

The NC-1 monomer of the invention is preferably longer than 20, 30, 40,50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, or 310 amino acidresidues and is able to dimerize or oligomerize. Further, it has atleast anti-fibrotic activity. In case the NC-1 monomer comprises theassociation domain, the hinge region and the zinc binding site/domain ofendostatin domain or the complete endostatin domain, it is preferredthat the NC-1 monomer is longer than 312 amino acid residues andcomprises even more preferred at least 315, 320, 330, 340, 350, 400, 500or even more amino acid residues.

In a further embodiment, the protein oligomer comprises at least twoendostatin domains of collagen 18 but can include also three, four, fiveor even more endostatin domains.

For instance, Fc-endostatin (FcE) as used in Example 11 consists of twoFc chains (connected by disulfide bonds), extended to two molecules ofendostatin each linked to a single Fc chain. Therefore, the two adjacentendostatin molecules become a dimer as a result of the Fc dimer. Anotherdimeric construct used in Example 11 comprised two endostatin domains ofcollagen 18. Each endostatin domain contained a single mutation atposition 7 in which glutamine was replaced by cysteine. Each endostatindomain was linked to a Fc region of an immunoglobulin, with anintervening enterokinase cleavage site. In this construct, both Fc andendostatin were separately dimerized by their corresponding disulfidebonds. Enterokinase digestion of this recombinant protein resulted in anFc dimer and an endostatin dimer. In another embodiment, the proteinoligomer or peptide oligomer comprises at least two N-terminal peptidesof the endostatin domain of collagen 18 but can include also three,four, five or even more of said N-terminal peptides.

The term “N-terminal peptide(s) of the collagen 18 endostatin domain” asused herein means a peptide from the amino-terminus of the endostatindomain of collagen 18. The definitions, explanations and embodiments asregards the NC-1 monomer and oligomerization thereof apply mutatismutandis to the N-terminal peptide(s) of the collagen 18 endostatindomain. The N-terminus of the endostatin domain of collagen 18 comprisesamino acid residues 1 to 132 of SEQ ID NO: 18 or 19 (corresponding tothe endostatin domain of collagen 18). Preferably, the N-terminalpeptide(s) of the collagen 18 endostatin domain is a endostatin peptide(without change of the amino acid sequence, in comparison to wild-type)or an endostatin-derived peptide as defined elsewhere herein.

In one embodiment, the N-terminal peptide of the collagen endostatindomain is or comprises an amino acid sequence from about amino acidresidue 1 to about amino acid residue 132 of SEQ ID NO: 18 or 19(endostatin domain of collagen 18).

The corresponding amino acid sequence of the endostatin domain of themurine collagen 18 is shown in SEQ ID NO: 18, whereas the correspondingamino acid sequence of the endostatin domain of the human collagen 18 isshown in SEQ ID NO: 19; see also FIG. 2 . The term “N-terminal peptideof the collagen 18 endostatin domain” as referred to in this applicationcomprises peptides located between about amino acid residues 1 (H;Histidine) and 132 (E; glutamic acid) of SEQ ID NO: 19, preferablybetween about amino acid residues 1 (H; Histidine) and 115 (P; Proline)of SEQ ID NO: 19, preferably between about amino acid residues 1 (H;Histidine) and 92 (G; Glycine), more preferably between about amino acidresidues 1 (H; Histidine) and 76 (D; Aspartic acid) of SEQ ID NO: 19,even more preferably between about amino acid residues 1 (H; Histidine)and 27 (R; Arginine) of SEQ ID NO: 19 or between amino acid residues 1(H; Histidine) and 25 (G; Glycine) of SEQ ID NO: 19. Examples forfurther N-terminal peptides of the collagen 18 endostatin domain areshown in Tjin et al., loc. cit., or in U.S. Pat. No. 7,524,811 orEP1668129 B1, incorporated herewith by reference. Preferably, theendostatin-derived peptide or endostatin peptide is about 10 to about 40amino acid residues in length, preferably about 15 to 35, preferablyabout 20 to 32, more preferably about 24, 25, 26, 27, 28, 29 or 30 aminoacid residues. Further preferred N-terminal peptides of the collagen 18endostatin domain are illustrated, in the following examples.

The N-terminal zinc-binding domain of endostatin or a synthetic peptidecorresponding to the N-terminal zinc-binding domain of endostatin isshown, for instance, in the amino acid sequences of SEQ ID NOs. 9 and10. It is encompassed by the present invention, that variants of theamino acid sequences of SEQ ID NOs. 9 and 10, e.g., shorter amino acidsequences of SEQ ID NOs. 9 and 10 can be used as well. For example, thepresent inventors have found that a peptide corresponding to positions 1to 13 of SEQ ID NO: 9 or positions 1 to 12 of SEQ ID NO: 10 can be usedas endostatin peptide in the protein or peptide oligomer. In addition,such a peptide can differ from the corresponding endostatin peptide orendostatin-derived peptide in one, two, three, four or even more aminoacid residues, while at least maintaining (or even exceeding) theanti-fibrotic activity (as described elsewhere herein) of thecorresponding endostatin peptide in the endostatin domain of NC-1. Inlight of this, it is important to maintain the Histidine amino acidresidues corresponding to positions 1, 3 and/or 11 of SEQ ID NOs. 9 or10 for the reasons set forth elsewhere herein. Moreover, replacement ofzinc by other similar divalent cations (i.e., copper) may result in morepotent N-terminal peptides of endostatin, endostatin and NC-1 domains ormonomers when compared with zinc.

The C-terminal endostatin polypeptides or peptides described in WO2011/050311 and Yamaguchi et al. (Sci. Transl. Med. 2012, 4, p.136ra71), i.e. peptides or polypeptides consisting of 40 consecutiveamino acid residues of amino acid residues 133-180 of SEQ ID NO: 2, 4 or13 in WO 2011/050311, and SEQ ID NO: 21 of the present application areexcluded from the scope of the present invention. It is of further notethat no anti-fibrotic activity could be shown for the N-terminalendostatin peptides El (amino acid residues 1 to 45) and E2 (amino acidresidues 71 to 115), in the publication by Yamaguchi et al., in contrastto the findings of the present inventions; see the following Examples.In light of this, the findings in the present application are even moresurprising.

The “NC-1 monomer of collagen 18”, “endostatin domain of collagen 18” orthe “N-terminal peptide of the collagen 18 endostatin domain” as definedherein can comprise additional protein domains or subunits, forinstance, the above-mentioned Fc domains of immunoglobulins, or proteintags, for example, His tags, c-myc tags, Flag tags or the like, whichcan be used, e.g., for purification and/or detection. As well known inthe art, protein tags are peptide sequences genetically grafted onto arecombinant protein. These tags can in one aspect be removable bychemical agents or by enzymatic means, such as proteolysis or inteinsplicing. Such tags are attached to the NC-1 monomer, endostatin domainof collagen 18 or N-terminal peptide of the collagen 18 endostatindomain as referred to herein. Affinity tags are appended to proteins sothat they can be purified from their crude biological source such as acell lysate using an affinity technique well known in the art. Theseinclude, for example, chitin binding protein (CBP), maltose bindingprotein (MBP), Fc domains of immunoglobulins orglutathione-S-transferase (GST). The poly(His) tag is a widely-usedprotein tag; it binds to metal matrices. Solubilization tags are used,especially for recombinant proteins expressed in chaperone-deficientspecies such as E. coli, to assist in the proper folding in proteins andkeep them from precipitating. These include, e.g., thioredoxin (TRX) andpoly-(NANP). Some affinity tags have a dual role as a solubilizationagent, such as MBP, and GST. Chromatography tags are used to alterchromatographic properties of the NC-1 monomer, endostatin domain ofcollagen 18 or N-terminal peptide of the collagen 18 endostatin domainto afford different resolution across a particular separation technique.Often, these consist of poly-anionic amino acids, such as the FLAG-tag.Epitope tags are short peptide sequences which are chosen becausehigh-affinity antibodies can be reliably produced in many differentspecies. These are usually derived from viral genes, which explain theirhigh immunoreactivity. Epitope tags include, for instance, V5-tag,c-myc-tag, and HA-tag. These tags are useful, e.g., for western blottingand immunoprecipitation experiments, although they also find use inprotein purification. Fluorescence tags are used to give visual readouton a protein. GFP and its variants are the most commonly usedfluorescence tags. More advanced applications of GFP include using it asa folding reporter (fluorescent if folded, colorless if not). Proteintags find many other usages, such as specific enzymatic modification(such as biotin ligase tags) and chemical modification (Flash tag). Thevarious tags can also be combined to produce multifunctionalmodifications of the NC-1 monomer, endostatin domain of collagen 18 orN-terminal peptide of the collagen 18 endostatin domain.

The NC-1 monomer of human collagen 18, endostatin domain of collagen 18or N-terminal peptide of the collagen 18 endostatin domain as definedherein can also comprise radioisotopes, e.g. ¹²⁴I, ¹²⁵I, ¹³¹I, Cu-64,Cu-67, Y-86, Zr-89, Y-90, Re-188, Ga-68; or radionuclides binding tochelates such as DTPA; toxins, e.g. Diphtheria toxin, or apoptosisinducing agents; or chemicals, e.g. chemotherapeutics such as taxols, orgemcitabine, which may be useful in improving and/or detecting theanti-fibrotic, anti-angiogenic and/or anti-tumorigenic activity of theprotein oligomer.

In other embodiments, the protein or peptide oligomer is pegylated.Pegylation is the process of covalent attachment of polyethylene glycol(PEG) polymer chains to another molecule, normally a drug or therapeuticprotein such as the protein or peptide oligomer as defined herein.Pegylation is routinely achieved by incubation of a reactive derivativeof PEG with the target macromolecule. The covalent attachment of PEG toa drug or therapeutic protein can “mask” the agent from the host'simmune system (reduced immunogenicity and antigenicity), increase thehydrodynamic size (size in solution) of the agent which prolongs itscirculatory time by reducing renal clearance. Pegylation can alsoprovide water solubility to hydrophobic drugs and proteins. Pegylationof compounds is well known in the art; see, e.g., Damodaran and Fee2010, European Pharmaceutical Review 15, 18.

The term “Fc region” or “Fc domain” as used herein means the fragmentcrystallizable region which is the tail region of an antibody orimmunoblobulin that interacts with cell surface receptors, i.e. Fcreceptors, and some proteins of the complement system. This propertyallows antibodies to activate the immune system. In IgG, IgA and IgDantibody isotypes, the Fc domain is composed of two identical proteinfragments, derived from the second and third constant domains of theantibody's two heavy chains; IgM and IgE Fc domains contain three heavychain constant domains (CH domains 2-4) in each polypeptide chain. TheFc domains of IgGs bear a highly conserved N-glycosylation site.Glycosylation of the Fc fragment is essential for Fc receptor-mediatedactivity. The N-glycans attached to this site are predominantlycore-fucosylated diantennary structures of the complex type. Inaddition, small amounts of these N-glycans also bear bisecting GlcNAcand α-2,6 linked sialic acid residues. Fusion of the Fc domain ofimmunoglobulins to proteins has been found to enhance the production andsecretion of the fusion proteins in mammalian cells (Lo et al., 1998,Protein Eng. 11, 495, Capon et al., 1989, Nature 337, 525). In addition,linking of angiogenesis inhibitors to an immunoglobulin Fc domain haveshown to increase the half life of said inhibitors (Capon et al. 1989,Nature 337, 525; Gordon et al., 2001, J. Clin. Oncol. 19, 843; Holash etal., 2002, Proc. Natl. Acad. Sci. USA 99, 11393). However, the Fc domaincan not only be used for purification, solubilization and/or detectionpurposes but alters advantageously the biological properties of theprotein or peptide oligomer, as set forth herein below and in thefollowing examples. In one embodiment, the one or more Fc domains can becleaved off by treatment with proteases, such as enterokinase orthrombin, if desired. Preferably, the Fc domain as referred to herein isfrom human IgG (Bergers and Javaherian Science 1999; Lee et al Clin CancRes 2008). As evident to those skilled in the art, in principle, any IgGisoform can be used to generate the oligomer of the invention. Evensub-fragments or single chains of the Fc domain of IgG can be used inorder to prolong the half life or oligomerization of the oligomerdescribed herein. The amino acid sequences of a mouse and human Fcdomain which can be used for the generation of an oligomer or a fusionprotein referred to herein, e.g. an Fc-NC-1 or NC-1-Fc fusion protein,or a Fc-endostatin or endostatin-Fc fusion protein, are shown in SEQ IDNOs: 5 and 6, respectively. Analogously, an N-terminal endostatinpeptide-Fc fusion protein or an Fc-N-terminal endostatin peptide can beused, in the protein oligomer or peptide oligomer. Put in other words:The Fc domain can be positioned N- or C-terminally, in the fusionprotein of the invention.

The protein oligomer or peptide oligomer, in an aspect, can bemanufactured by chemical synthesis or recombinant molecular biologytechniques well known to the person skilled in the art; see, e.g.,Sambrook et al., Molecular cloning: a laboratory manual/Sambrook,Joseph; Russell, David W.—. 3rd ed.—New York: Cold Spring HarborLaboratory, 2001.

For example, the protein oligomer or peptide oligomer can be generatedby a method, comprising (a) culturing a host cell comprising a nucleicacid sequence encoding the protein oligomer or peptide oligomer,preferably under serum-free conditions, (b) obtaining from the host cellof step (a) the protein oligomer or peptide oligomer, and, optionally,(c) storing the protein oligomer or peptide oligomer, preferably underserum-free conditions. It has been found by the present inventors, thatoligomeric NC-1 such as the NC-1 trimer is susceptible to degradation ifkept in serum or cell culture media for longer periods of time, even at4° C. Therefore, it is advantageous to produce and keep the proteinoligomer or peptide oligomer under serum-free conditions. Alternatively,a polynucleotide encoding an NC-1 monomer of collagen 18, endostatindomain of collagen 18 or an N-terminal peptide of the endostatin domainof collagen 18 can be expressed under suitable conditions, in anappropriate host cell. Assembly of the mentioned NC-1 monomer,endostatin domain of collagen 18 or N-terminal endostatin peptide todimers or oligomers occurs within the cell. Subsequently, the dimer oroligomer can be isolated and/or purified by methods known in the art(see, e.g., Sambrook, Molecular Cloning A Laboratory Manual, Cold SpringHarbor Laboratory (2001) N.Y. and Ausubel, Current Protocols inMolecular Biology, Green Publishing Associates and Wiley Interscience,N.Y. (1994)). For example, the protein oligomer or peptide oligomer canbe obtained by conventional purification techniques from, e.g., a hostcell lysate including, but not limited to, affinity chromatography, ionexchange chromatography, size exclusion chromatography and/orpreparative gel electrophoresis. Alternatively, the mentioned NC-1monomer, endostatin domain of collagen 18 or N-terminal endostatinpeptide can be assembled to dimers or oligomers in vitro, afterisolation and/or purification of the NC-1 monomer, endostatin domain ofcollagen 18 or N-terminal endostatin peptide from the cell.

In one embodiment, the protein or peptide oligomer binds to Fibronectin.In addition, the oligomer can bind to VEGF, preferably VEGF-A, MMP-2,MMP-9 and/or integrin alpha 5 beta 1. Said binding of the protein orpeptide oligomer of the invention to Fibronectin, VEGF, MMP-2, MMP-9and/or integrin alpha 5 beta 1 can be determined by methods known in theart such as immunoprecipitation, ELISA assays or Biacore.

In a further embodiment of the protein oligomer or peptide oligomer, theNC-1 monomer of human collagen 18 comprises an oligomerization domain, ahinge region and/or an endostatin domain or fragments of said endostatindomain. In some embodiments, the NC-1 monomer of human collagen 18comprises an oligomerization domain, a hinge region and a completeendostatin domain. In other specific embodiments, the NC-1 monomer ofhuman collagen 18 comprises an oligomerization domain, a hinge regionand a fragment of the endostatin domain. The fragment of the endostatindomain is preferably an N-terminal fragment of the endostatin domain,more preferably an N-terminal peptide of the endostatin domain or apeptide derived from the N-terminus of the endostatin domain of collagen18. The fragment of the endostatin domain comprises preferably at leastone N-terminal endostatin peptide as defined herein, in the NC-1monomer. The fragments of said endostatin domain can comprise also two,three, four or even more N-terminal endostatin peptides as definedherein, in the NC-1 monomer.

In another preferred embodiment of the protein oligomer or peptideoligomer, the hinge region is interposed between the oligomerizationdomain and the endostatin domain or fragment(s) of said endostatindomain. Preferably, the hinge region is located between theoligomerization domain and the endostatin domain or fragment(s) thereof,in the NC-1 monomer as referred to herein. The domain arrangement withinthe NC-1 monomer of human collagen 18 is preferably oligomerizationdomain-hinge region-endostatin domain or fragment(s) of said endostatindomain, or endostatin domain or fragment(s) of said endostatindomain-hinge region-oligomerization domain.

Optionally, the hinge region within the NC-1 monomer of human collagen18 may comprise one or more recombinant protease cleavage sites, inaddition or alternatively to the endogenous MMP protease cleavage sitesof the hinge region. Such a recombinant protease cleavage site can be,for instance, an enterokinase or thrombin cleavage (Bergers andJavaherian; Lee et al.; loc. cit.; Lo et al., 1998, Protein Engineering11(6), 1998, p. 495-500). Said publications further describe appropriatelinker regions that can be used for introducing the mentioned cleavagesite(s), such as, e.g., poly-Glycine linkers and the like. Cleavage bythe respective protease allows for, e.g., the release of the endostatindomain(s) of the protein oligomer or peptide oligomer.

In alternative embodiments, the NC-1 monomer as referred to herein lacksthe hinge region.

In another embodiment of the protein oligomer or peptide oligomer, theNC-1 monomer of collagen 18, the endostatin domain of collagen 18 or theN-terminal peptide of the collagen 18 endostatin domain furthercomprises an RGD motif and/or PHSRN motif of Fibronectin, preferably ina fusion protein. Preferably, the NC-1 monomer of collagen 18, theendostatin domain of collagen 18 or the N-terminal peptide of thecollagen 18 endostatin domain comprise an RGD motif and a PHSRN motif ofFibronectin.

For instance, SEQ ID NOs. 11 and 12 provide amino acid sequencescomprising the RGD motif and surrounding amino acid residues importantfor binding of Fibronectin to integrins. Briefly, Fibronectin isrecognized by integrins alpha 5 beta 1 and alpha V beta 3. The primarysequence motif of fibronectin for integrin binding is a tripeptide,Arg-Gly-Asp (RGD), located on the loop connecting the force-bearing G-and F-strands of FN-III10. Further involved in integrin binding ofFibronectin is the Pro-His-Ser-Arg-Asn (PHSRN) motif which resides inthe ninth domain of type III fibronectin.

The corresponding amino acid sequences of murine and human Fibronectin(FN) are shown, e.g., in accession numbers NP _034363.1 and NP_997647.1, respectively. The domain structure of human FN can bederived, e.g., from the publication by Wijelath et al. 2006, Circ. Res.99, 853-860. Preferably, the RGD motif of Fibronectin comprises orconsists of SEQ ID NO. 11, 12 or 17.

Preferably, the endostatin peptide or endostatin-derived peptide islocated at the N-terminal end of the fusion protein and the RGD motifand/or PHSRN motif of Fibronectin is located at the C-terminal end.

Preferably, such a fusion protein comprises an amino acid sequence asshown in SEQ ID NO: 7 or 13.

In another preferred embodiment, the fusion protein further comprises anFc domain or an artificial oligomerization domain as defined herein.Preferably, the fusion protein with an artificial oligomerization domaincomprises an amino acid sequence as shown in SEQ ID NO: 15. Preferably,the fusion protein comprises an Fc domain and an artificialoligomerization domain as defined herein

Based on previous experimental data in WO 2013/026913, the presentinventors hypothesized that oligomeric NC-1 may elicit its effects viaFibroncectin (FN). In addition to this, it has been found in thefollowing Examples that oligomeric NC-1 and oligomeric endostatin bindto, VEGF and the matrix metalloproteinases MMP-2/MMP-9 which areimportant players in remodeling of extracellular matrix, in developmentof fibrosis, cancer progression and metastasis. Moreover, they found inWO 2013/026913 that FN is significantly down-regulated in tumors thatbecome resistant to oligomeric NC-1 (Fc-Endostatin) after prolongedexposure, i.e. four serial in-vivo passages. Therefore, they postulatedthat loss of FN might constitute a key mechanism of inherent andacquired resistance to oligomeric NC-1. To proof this concept, a minimalpeptide sequence has been engineered that mimics the key effects of theendostatin (ED)—Fibronectin complex. To this end, the inventors firstselected the most active motif in the entire ED-domain consisting of a27 amino acid-NH₂-terminal region (Tjin Tham Sjin et al. 2005, CancerRes. 65, 3656-63). Data by the present inventors indicated that thisregion itself may be capable of binding to VEGF and that the Histidines(Zinc binding domain) in this peptide sequence may be critical for VEGFbinding. This is conceivable, because a mutated peptide in which theHistidines were replaced by Alanine residues failed to compete withVEGF-ED-dimer (Fc-Endostatin) binding. On the other hand, Fibronectincontains two active motifs that are critical for its binding to ITGA5B1,i.e. a PHSRN- and a RGD-dependent motif. In order to mimic thephysiological complex of oligomeric NC-1 and FN that mediated integrinsignaling and other properties of the NC-1-ED, the inventors fused thesetwo critical motifs, i.e. the above-mentioned most active motif in theNC-1-ED domain and the integrin-binding motif of Fibronectin comprising“RGD” and surrounding amino acid residues important for binding, andgenerated chimeric (or hybrid) fusion proteins called “Superstatins”.For each fusion protein, a mouse and a human equivalent was designed.Using the murine (C57BL6) LLC (Lewis lung carcinoma) lung cancer model,the inventors were able to show the efficacy of the murine Superstatinpeptide to potently inhibit tumor growth. In addition, Superstatinsignificantly prolonged survival as compared to control. In contrast,the FN-Motif alone showed no significant improvement in prevention oftumor growth.

The corresponding amino acid sequence for the murine (m) Superstatin isshown in SEQ ID NO: 7, whereas the corresponding amino acid sequence forthe human (h) Superstatin is shown in SEQ ID NO: 13. Superstatins arelikely monomers. SEQ ID NO: 15 shows a variant of the human Superstatinamino acid sequence which is able to dimerize, due to the substitutionof Glutamine at position 7 in SEQ ID NO: 13 by Cysteine. Additionalconstructs containing the PHSRN instead of the RGD motif of FN, as wellas constructs facilitating dimerization of the Superstatin via disulfidebounds or Fc regions will be prepared and evaluated with respect toanti-fibrotic activity.

In a further embodiment of the protein oligomer or peptide oligomer, theNC-1 monomer of human collagen 18, the endostatin domain of collagen 18or the N-terminal peptide of the collagen 18 endostatin domain comprisesa native or heterologous oligomerization domain.

Preferably, the native oligomerization domain is a non-triple helicaltrimerization domain of human collagen 18.

Preferably, the heterologous oligomerization domain is anoligomerization domain selected from the group consisting of an Fcdomain and an artificial oligomerization domain.

The oligomerization domain as referred to herein can comprise anon-triple helical trimerization domain of human collagen 18 (, i.e. theassociation domain), an Fc domain or an artificial oligomerizationdomain. The oligomerization domain comprises in one aspect a nativeoligomerization domain, i.e. a non-triple helical trimerization domainof human collagen 18 which is responsible for trimerization of the threechains of the NC-1 domain. In another aspect, it comprises aheterologous oligomerization domain, e.g. an Fc domain from an antibodyor immunoglobulin. The Fc domain confers a dimeric structure on the NC-1monomer, endostatin domain or the N-terminal peptide of the endostatindomain as defined herein since the Fc domain is a dimer itself. In athird aspect, it comprises an artificial oligomerization domain, forexample, point mutations to cysteins resulting in disulfide bridgesbetween two monomers which replaces structurally and functionally theassociation domain as found in the natural human NC-1 referred to above,or is used in addition to said association domain or Fc domain. Othermeans and methods for dimerization or oligomerization have beendescribed elsewhere herein and are known in the art including, e.g.,coiled coils, leucine zipper, CovX body technology etc. and arecomprised by the term “heterologous oligomerization domain” or“artificial oligomerization domain” as used herein. It is alsoencompassed by the scope of the invention, that the oligomerizationdomain of the protein oligomer comprises a non-triple helicaltrimerization domain of human collagen 18 and a Fc domain. Further, itcan comprise an artificial oligomerization domain and a Fc domain. Forinstance, one of the dimeric constructs used in Example 11 comprised twoendostatin domains of collagen 18. Each endostatin domain contained asingle mutation at position 7 in which glutamine was replaced bycysteine. Each endostatin domain was linked to a Fc region of animmunoglobulin, with an intervening enterokinase cleavage site. In thisconstruct, both Fc and endostatin were separately dimerized by theircorresponding disulfide bonds. Enterokinase digestion of thisrecombinant protein resulted in an Fc dimer and an endostatin dimer.

Preferably, the Fc domain is from IgG or other immunoglobulin isoformsas well as other scaffold constructs providing oligomerization andlonger half life described in the art; see, e.g., Lo et al., ProteinEngineering 1998,11,495. A murine Fc domain is shown, for example, inSEQ ID NO: 5. More preferably, the Fc domain is from a human IgG, evenmore preferred from human IgG1. Particularly preferred, the human Fcdomain comprises or consists of an amino acid sequence as shown in SEQID NO: 6 or SEQ ID NO: 24.

In another preferred embodiment, the Fc domain is a “knobs-into-holes”(KiH) engineered Fc domain. Knobs-into-holes is a well-validatedheterodimerization technology for the third constant domain of anantibody. Basically, the concept relies on modifications of theinterface between the two CH3 domains where most interactions occur. Abulky residue is introduced into the CH3 domain of one antibody heavychain and acts similarly to a key. In the other heavy chain, a “hole” isformed that is able to accommodate this bulky residue, mimicking a lock.The resulting heterodimeric Fc-part can be further stabilized byartificial disulfide bridges. During the process of optimizing theheterodimerization interface, various rational designs, including stericcomplementarity, KiH, disulfide bonds and salt bridges juxtaposingoppositely charged residues on either side of the CH3 domain, wereevaluated and ultimately optimized using a phage display library.Correct heavy chain association with heterodimerization yields above 97%can be achieved by introducing six mutations: S354C, T366W in the “knob”heavy chain and Y349C, T366S, L368A, Y407V in the “hole” heavy chain(Klein et al., MAbs. 2012 Nov 1; 4(6): 653-663; Ridgway et., ProteinEng. 1996 Jul;9(7):617-21). In addition, properties of antibodies withKiH mutations such as (thermal) stability, FcyR binding and effectorfunctions (e.g., ADCC, FcRn binding) and pharmacokinetic (PK) behaviorare not affected. The noncovalent interactions, along with disulfidebridges in the hinge region, drive assembly toward heterodimer formationand minimize combinatorial heterogeneity. The production of NC-1 usingconventional approaches suffers from low protein yields. Further, alsoproduction of NC-1 fused to Fc is no trivial task because of theformation of a number of different aggregations. Such heterogeneity isunwanted in pharmaceutical compositions, as appreciated by those skilledin the art. The KiH technology can be used to produce NC-1 as a monomerand avoids the formation of such heterogeneous aggregations (such as amixture of NC-1 dimers, trimers, tetramers, pentamers and the like).Suitable KiH-engineered Fc domains are depicted, e.g., in SEQ ID NOs.25, 26, 28 and 30. For example, SEQ ID NO: 25 shows the amino acidsequence of the human IgG1 Fc with the “knob” mutations S354C/T366W, andSEQ ID NO: 26 depicts the amino acid sequence of the human IgG1 Fc withthe “hole” mutations Y349C/T366S/L368A/Y407V.

SEQ ID NO: 27 shows the amino acid sequence of a fusion proteincomprising human NC-1 fused via an enterokinase cleavage site and alinker to the human IgG1 Fc with “knob” mutations (S354C/T366W) (from N-to C-terminus). This fusion protein is able to heterodimerize with thehuman IgG1 Fc with “hole” mutations Y349C/T366S/L368A/Y407V (SEQ ID NO:28). Such a heterodimer is illustrated in FIG. 14B. Cleavage of theheterodimer by enterokinase results in the generation of NC-1 monomer.The NC-1 monomer can then be used for the generation of NC-1 dimers orNC-1 trimers. Evidently, endostatin can be used, instead of NC-1, ifmonomeric endostatin shall be produced.

SEQ ID NO: 29 shows the amino acid sequence of a fusion proteincomprising human IgG1 Fc with “knob” mutations (S354C/T366W) fused via alinker and an enterokinase site to human NC-1 (from N- to C-terminus).This fusion protein is able to heterodimerize with the human IgG1 Fcwith “hole” mutations Y349C/T366S/L368A/Y407V (SEQ ID NO: 30). Such aheterodimer is illustrated in FIG. 14A. Cleavage of the heterodimer byenterokinase results in the generation of NC-1 monomer. Obviously,endostatin can be used, instead of NC-1, if monomeric endostatin shallbe produced.

“Knobs-into-holes” (KiH)-engineered Fc domains are particularly usefulfor the production of NC-1 monomers or endostatin monomers, for theabove-indicated reasons. Accordingly, it is preferred thatKiH-engineered Fc domains of human immunoglobulins, more preferablyKiH-engineered Fc domains of human IgG1 be used for the production ofthe protein oligomer, peptide oligomer or fusion protein of theinvention.

In another embodiment, a NC-1 monomer of collagen 18 and a Fc region ofan immunoglobulin is expressed together with a Fc region of animmunoglobulin in a cell to avoid uncontrolled aggregation of the NC-1.This approach results in the formation of a Fc-Fc dimer with one Fcconnected to the NC-1 monomer. If a protease (such as thrombin orenterokinase) cleavable linker is interposed between the Fc and NC-1monomer, the NC-1 monomer can be released from the heterodimer uponcleavage with the respective protease. A similar approach can be usedfor the generation of an endostatin monomer.

In a still further embodiment, it can be appropriate to omit or mutatethe native N-terminal association region in the NC-1 domain so that NC-1cannot oligomerize any longer via its natural association region. Forexample, a series of recombinant NC-1 domains can be produced in whichthe native association region is subsequently reduced by, e.g., 3, 5 or10 amino acid residues. Then they are tested for their oligomerizationproperties in order to identify NC-1 variants that are no longer able tooligomerize via their native association region. For testing theoligomerization properties of said variants, Western blot analysis,immunoprecipitation, SDS-PAGE, chromatographic methods or other methodswell known in the art can be utilized. The recombinant polypeptidesgenerated by the above-indicated method can be used to produce proteinoligomers or fusion proteins of the invention which can then further betested for their biological activity as defined herein. In anotherembodiment, amino acid residues required for the association areidentified by structural analyses (e.g. computational or 3D structuralanalysis like SAPIN) and then mutated accordingly by recombinant methodsknown in the art (see Sambrook and Russell, 2001, loc. cit.) so that theNC-1 cannot oligomerize any longer. In another embodiment, the nativeN-terminal association region in the NC-1 domain can be omittedcompletely. If a NC-1 domain without functional association region isfused to a Fc region, such a monomer can be used for the production of aNC-1 dimer wherein dimerization is mediated via the Fc region. Theprotein oligomer of the invention can comprise at least one, or two ormore of NC-1 monomers of human collagen 18 with such alterations ordeletions in the native N-terminal association region, as definedelsewhere herein.

The oligomerization domain of the NC-1 monomer, endostatin domain or theN-terminal peptide of the endostatin domain can be a Fc domain of animmunoglobulin, preferably a Fc domain from IgG1, or a KiH-engineered Fcdomain from IgG1, as set forth above. The protein oligomer or peptideoligomer can also contain two, three or even more Fc domains. In oneaspect, the Fc domain(s) may be cleaved off the protein oligomer orpeptide oligomer, if desired. For instance, an artificial proteasecleavage site such as an enterokinase or a thrombin cleavage site can beinterposed between the NC-1 monomer or endostatin domain and the Fcdomain(s) in the protein oligomer or peptide oligomer, for example, viaa corresponding (poly)peptide linker (see, e.g., Bergers and Javaherian;Lee et al.; loc. cit.; Lo et al., 1998, Protein Engineering 11(6), 1998,p. 495-500). Upon cleavage by the respective protease, the oligomer isreleased from the Fc domain(s).

The Fc domain(s) can also be used for purification and/or detection. Inaddition, the Fc domain alters the biological properties of the proteinoligomer, such as half-life extension in circulation and improvement ofbiological activity, preferably improvement of the anti-fibrotic,anti-angiogenic activity and/or anti-tumor activity. For example, it hasbeen found that an Fc-endostatin fusion protein is able to bindFibronectin as a dimer, whereas endostatin monomer does not, asdemonstrated in the following Examples. Moreover, Fc-endostatin shows alonger half life than endostatin.

In a further preferred embodiment of the protein oligomer or peptideoligomer, the artificial oligomerization domain comprises a singlemutation at position 7 of the endostatin domain in which glutamine isreplaced by cysteine. Preferably, the NC-1 monomer, the endostatindomain or N-terminal peptide of the endostatin domain as defined hereincomprises in some aspects a single mutation of glutamine to cysteine atposition 7 of the endostatin domain. For example, it has been found thata recombinantly introduced enterokinase cleavage site between the Fcdomain and endostatin domain in a fusion protein results in theformation of a dimer upon enterokinase cleavage because of disulfidebond formation between adjacent C7 residues in the endostatin domains;see Kuo 2001, JCB 152, 1233 and the following Examples. As set forthabove, NC-1 trimer and endostatin dimers have distinct properties, incomparison to the endostatin monomer. The above mutation at position 7(glutamine to cysteine) can also be introduced in the N-terminal peptideof endostatin which has been shown to represent the anti-tumor domain ofendostatin (Tjin et al. 2005, Cancer Res 65, 3656). The oligomerizationof the N-terminal peptide of the endostatin domain can be achieved byeither artificial dimerization as described above or simply byrecombinant fusion to the Fc moiety without a mutation in position 7. Anexample for a fusion protein comprising said mutation at position 7mediating dimerization is shown in SEQ ID NO: 15.

In another preferred embodiment of the protein oligomer or peptideoligomer, the recombinant protease cleavage site within the hinge regionis an enterokinase or thrombin cleavage site.

The cleavage of the protein oligomer or peptide oligomer with theenterokinase or thrombin results in the release of the endostatindomains from the protein oligomer or peptide oligomer.

In a further preferred embodiment of the protein oligomer or peptideoligomer, the NC-1 monomer as defined herein contains only a proteasecleavage site naturally occurring within the hinge region, i.e. it doesnot comprise a recombinant protease cleavage site. In this case, thehinge region can be cleaved, e.g. by MMPs, as set forth elsewhereherein, in order to release, e.g., the endostatin domain(s), from theNC-1 monomer. In another aspect, these naturally occurring proteasecleavage sites in the hinge region of the NC-1 monomer can be mutated sothat NC-1 monomer is no longer cleaved by said proteases. In this way,e.g., the half-life, anti-fibrotic, anti-angiogenic and/or anti-tumoractivity of the protein oligomer may still be improved.

In another preferred embodiment of the protein oligomer or peptideoligomer, the oligomer is a dimer or a trimer. However, encompassed bythe protein oligomer or peptide oligomer are also tetramers or pentamersor oligomers with even more NC-1 monomers, endostatin domains orN-terminal peptides of the endostatin domain, as defined herein.

A pharmaceutical composition comprising the protein oligomer or peptideoligomer as pharmaceutical active compound can be used for non-human or,preferably, human therapy of fibrosis or fibrosis-associated diseases,in a therapeutically effective dose. The pharmaceutical composition ofthe invention can be also used for therapy of a vascular endothelialgrowth factor (VEGF)-related disease or a matrix metalloproteinase(MMP)-related disease. The “subject” as referred to herein is preferablya human suffering from fibrosis or a fibrosis-associated disease, avascular endothelial growth factor (VEGF)-related disease or a matrixmetalloproteinase (MMP)-related disease.

Preferably, the fibrosis-associated disease is selected from the groupconsisting of: fibrosis of the skin, preferably scleroderma; keloid orkeloid scar; hypertrophic scar; morphea; fibrosis as a result ofgraft-versus-host disease; subepithelial fibrosis; endomyocardialfibrosis; uterine fibrosis; myelofibrosis; retroperitoneal fibrosis;nephrogenic systemic fibrosis; scarring after surgery; asthma;cirrhosis/liver fibrosis; fibrosis as a result of aberrant woundhealing; glomerulonephritis; multifocal fibrosclerosis;radiation-induced fibrosis (as an example for stimulation-inducedfibrosis), preferably radiation-induced pneumonitis or radiation-inducedlung fibrosis; chemotherapy-induced or drug-induced fibrosis, e.g., as aresult of mTOR or EGFR kinase inhibition; usual or idiopathic pulmonaryfibrosis (as an example for idiopathic fibrosis); fibrosis as the resultof autoimmune diseases, e.g., Lupus, intra-tumoral- andcancer-associated fibrosis/fibrogenesis, organ fibrosis-followed chronicinflammation (e.g., via viral stimulus or transplantation); organfibrosis as the endstage of chronic kidney diseases, long term dialysis,or diabetes mellitus.

Preferably, the vascular endothelial growth factor (VEGF)-relateddisease is selected from the group consisting of: benignpathophysiological conditions depending on deregulation of the VEGFlevels such as wet macular degeneration, endometriosis, bronchial asthmaand diabetes mellitus, enhanced VEGF-induced vascular permeability(e.g., enhanced permeability after irradiation of brain tissue,“radionecrosis”), alterations of vaso-tonus (e.g. hypertension),rheumatoid arthritis, as well as malignant VEGF-related diseases such asrenal cell cancer and other VEGF-addicted tumors, VEGF-dependentdevelopment of ascites, VEGF-dependent suppression of immune system,e.g. recruitment and microenvironmental education of bone marrow-derivedcells (BMDC), myeloid derived suppressor cells (MdSC), or immaturedendritic cells.

Preferably, the matrix metalloproteinase (MMP)-related disease isselected from the group consisting of: benign and malignant diseaseswhere MMP activation contributes to the pathophysiology, e.g.,activation of MMPs during the process of local tumor invasion and cancermetastasis inherently evident in tumors with high local therapy failurerates such as glioblastoma, pancreatic cancer, lung cancer, as well asacquired enhanced MMP activation as the function of therapy inducedselection pressures (e.g. tumor hypoxia and fibrosis post radiotherapy),overt immune reaction in autoimmune diseases and chronic inflammatorydiseases.

In an aspect, the protein oligomer or peptide oligomer can be present inliquid or lyophilized form. In an aspect, the protein oligomer orpeptide oligomer can be present together with glycerol, proteinstabilizers (e.g., human serum albumin (HSA)) or non-proteinstabilizers.

The protein oligomer or peptide oligomer is the active ingredient of thepharmaceutical composition or medicament (both terms are usedinterchangeably), and is in one aspect, administered in conventionaldosage forms prepared by combining the drug with standard pharmaceuticalcarriers according to conventional procedures. These procedures mayinvolve mixing, granulating, and compressing, or dissolving theingredients as appropriate to the desired preparation. It will beappreciated that the form and character of the pharmaceutical acceptablecarrier or diluent is dictated by the amount of active ingredient withwhich it is to be combined, the route of administration, and otherwell-known variables.

The carrier(s) must be acceptable in the sense of being compatible withthe other ingredients of the formulation and being not deleterious tothe recipient thereof. The pharmaceutical carrier employed may include asolid, a gel, or a liquid. Exemplary of solid carriers are lactose,terra alba, sucrose, talc, gelatine, agar, pectin, acacia, magnesiumstearate, stearic acid and the like. Exemplary of liquid carriers arephosphate buffered saline solution, syrup, oil, water, emulsions,various types of wetting agents, and the like. Similarly, the carrier ordiluent may include time delay material well known to the art, such asglyceryl mono-stearate or glyceryl distearate alone or with a wax. Saidsuitable carriers comprise those mentioned above and others well knownin the art, see, e.g., Remington's Pharmaceutical Sciences, MackPublishing Company, Easton, Pa.

The diluent(s) is/are selected so as not to affect the biologicalactivity, preferably, anti-fibrotic activity, anti-angiogenic activity,anti-invasive/anti-metastatic activity, reducing vascular permeabilityactivity, anti-inflammatory and/or anti-tumorigenic activity of thepharmaceutical composition. Examples of such diluents are distilledwater, physiological saline, Ringer's solutions, dextrose solution, andHank's solution. In addition, the pharmaceutical composition orformulation may also include other carriers, adjuvants, or non-toxic,non-therapeutic, non-immunogenic stabilizers and the like.

The protein oligomer or peptide oligomer is preferably formulated as apharmaceutical composition which can be administered by standard routes.Generally, the pharmaceutical composition may be administered by thetopical, transdermal, intraperitoneal, intracranial/intrathecal,intravitreal, intracerebroventricular, intracerebral, intravaginal,intrauterine, oral, rectal or parenteral (e.g. intravenous, intraspinal,subcutaneous or intramuscular) route.

Preferably, the protein oligomer or peptide oligomer is administeredintravenously, subcutaneously, intracranial/intrathecal, intravitreal,or intraperitoneally.

A therapeutically effective dose refers to an amount of the proteinoligomer or peptide oligomer to be used in a pharmaceutical compositionwhich prevents, ameliorates or treats the symptoms accompanying thedisease referred to in this specification. Therapeutic efficacy andtoxicity of the compound can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., ED50 (thedose therapeutically effective in 50% of the population) and LD50 (thedose lethal to 50% of the population). The dose ratio betweentherapeutic and toxic effects is the therapeutic index, and it can beexpressed as the ratio, LD50/ED50.

The dosage regimen will be determined by the attending physician andother clinical factors. As is well known in the medical arts, dosagesfor any one patient depends upon many factors, including the patient'ssize, body surface area, age, the particular compound to beadministered, sex, time and route of administration, general health, andother drugs being administered concurrently. Progress can be monitoredby periodic assessment.

Preferably, the protein oligomer or peptide oligomer is administered ina concentration from about 1 to 100 mg/kg. More preferably, theconcentration is from about 5 to 75 mg/kg or from about 10-50 mg/kg,most preferably about 15 mg/kg. Even more preferred, the proteinoligomer or peptide oligomer is administered at a concentration of 0.1-1mg/kg/day.

The medicament or pharmaceutical composition referred to herein isadministered at least once in order to treat or ameliorate or preventthe disease recited in this specification. However, the said medicamentmay be administered more than one time, e.g., two, three, four, five,six times or even more frequently.

Specific pharmaceutical compositions are prepared in a manner well knownin the pharmaceutical art and comprise at least one active compoundreferred to herein above in admixture or otherwise associated with apharmaceutically acceptable carrier or diluent. For making thosespecific pharmaceutical compositions, the active compound(s) willusually be mixed with a carrier or the diluent. The resultingformulations are to be adapted to the mode of administration. Dosagerecommendations shall be indicated in the prescribers or usersinstructions in order to anticipate dose adjustments depending on theconsidered recipient.

The pharmaceutical composition may in a further aspect of the inventioncomprise drugs in addition to the protein oligomer which are added tothe medicament during its formulation. For example, it can be usedtogether with angiostatin, in a combination regimen. Further,combinations with recently approved modulators of fibrosis such asVEGF/PDFG RTKi (e.g. Nindetanib), specific and non-specific inhibitorsof TGF-beta-signaling (Perfinidone) and modulators of integrin signaling(cilengitide, or anti alphaV abituzumab) or inflammation (leukocyteinfiltration, cytokine inhibitors, antibodies against subpopulations)are envisaged, in another aspect. Medicaments for the therapy of avascular endothelial growth factor (VEGF)-related disease which can beused in addition to the protein oligomer or peptide oligomer include,for example, other modulators of vascular permeability (e.g. enhancedpermeability after irradiation of brain tissue, “radionecrosis”) andvaso-tonus (e.g. endothelin antagonists macitentan, AT1/ACE inhibitors),β2-sympathomimetics and corticoids in asthma, immune-suppressants inchronic inflammatory/auto-immune diseases, chemotherapy and radiotherapyfor different VEGF dependent tumors and ascites, kinase inhibitors usede.g. in renal cell cancer (mTORi e.g., RAD001, multikinase inhibitorspazopanib/suitinib/axitinib, immune modulators e.g. checkpointinhibitors anti PD-1/PD-11). Medicaments for the therapy of a matrixmetalloproteinase-related disease which can be used in addition to theprotein oligomer or peptide oligomer include, for example, locallyinvasive tumors with high loco-regional therapy failure rates treatedwith radio-(chemo)-therapy such as glioblastoma, pancreatic cancer,anti-inflammatory and immunosuppressive therapy (anti-TNF alphaantibodies/infliximab, mycophenolic acid, cyclophosphamide etc.), tumorinvasion or pseudoprogression after cancer treatment e.g.anti-angiogenic therapy in recurrent glioma, treatments of metastaticdiseases with high MMP-2/MMP-9 activity such as breast cancer (i.e.hormonal therapy tamoxifen, Trastuzumab in HER2+ disease,chemotherapies).

Thus, in preferred embodiments of the protein oligomer or peptideoligomer, said oligomer further comprises angiostatin (U.S. Pat. No.8,206,718). In specific embodiments, the angiostatin is anFc-angiostatin or angiostatin-Fc fusion protein, preferably human fusionprotein.

It is to be understood that the formulation of a pharmaceuticalcomposition takes place under GMP standardized conditions or the like inorder to ensure quality, pharmaceutical security, and effectiveness ofthe medicament.

As evident from the above, it is preferred that the protein oligomer,peptide oligomer and fusion protein is or is composed of humansequences.

The invention further pertains to a protein oligomer comprising (i) atleast two NC-1 monomers of human collagen 18 or (ii) at least twoendostatin domains of collagen 18 or (iii) at least two N-terminalpeptides of the collagen 18 endostatin domain, for use for detectingand/or diagnosing fibrosis or a fibrosis-associated disease, a vascularendothelial growth factor (VEGF)-related disease or a matrixmetalloproteinase-related disease.

The definitions and embodiments provided as regards the medical uses ofthe protein oligomer or peptide oligomer apply mutatis mutandis to thediagnostic application and uses of the invention.

The term “detecting” as utilized herein means to discover or ascertainthe existence or presence of fibrosis or a fibrosis-associated disease,a vascular endothelial growth factor (VEGF)-related disease or a matrixmetalloproteinase-related disease, in a subject.

The term “diagnosing” as referred to in this description means torecognize fibrosis or a fibrosis-associated disease, a vascularendothelial growth factor (VEGF)-related disease or a matrixmetalloproteinase-related disease, in a subject by examination.Diagnosis as used herein is to be understood as medical diagnosis whichrefers to both the process of attempting to determine or identify apossible disease and diagnosis in this sense can also be termed(medical) diagnostic procedure, and to the opinion reached by thisprocess also being termed (medical) diagnostic opinion.

Detecting and diagnosing of fibrosis or a fibrosis-associated disease, avascular endothelial growth factor (VEGF)-related disease or a matrixmetalloproteinase-related disease, in a subject can be carried out bymethods known in the art such as computer tomography (e.g., highresolution computed tomography), ultrasound, blood analysis (e.g. bloodgas analysis, acid-basic balance), analysis of biological markers suchas proteinases (MMPs), growth factors and/or cytokines, histopathology(collagen deposition, inflammatory response markers), clinical tests(organ function, e.g. restrictive lung diseases test FEV1 etc., organdysfunction), cellular tests, invasive (surgical biopsy) andnon-invasive tests, other invasive and non-invasive examinations such asMRI, PET/CT and the like, or a combination thereof (see, e.g., Raghu etal. 2011, Am. J. Respir. Crit. Care Med. 183, p. 788-824).

For example, the accuracy of diagnosis of idiopathic pulmonary fibrosis(IPF) increases with clinical, radiologic, and histopathologiccorrelation and can be accomplished with a multidisciplinary discussionamong experienced clinical experts in the field.

For detecting and/or diagnosing fibrosis or a fibrosis-associateddisease, a vascular endothelial growth factor (VEGF)-related disease ora matrix metalloproteinase-related disease, the NC-1 monomers of humancollagen 18, the endostatin domain of collagen 18 or the N-terminalpeptides of the collagen 18 endostatin domain can be labeled withradioisotopes, radionuclides binding to chelates such as DTPA,fluorescent proteins or other labels described elsewhere herein.

For example, the human Superstatin peptide (SEQ ID NO: 13) can beconjugated to the complexing agent1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (also known asDOTA) providing the ability to conjugate the peptide with, e.g.,radionuclides such as Gallium (⁶⁸Ga) for non-invasive imaging (Positronemission tomography, PET). In-vivo PET-Imaging evaluating the potentialof Superstatin-DOTA as agent for diagnosing a fibrosis-associateddisease, a vascular endothelial growth factor (VEGF)-related disease ora matrix metalloproteinase-related disease, is envisioned.

Thus, preferably, the human Superstatin peptide (SEQ ID NO: 13) isconjugated to the complexing agent1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (also known asDOTA).

The invention also relates to fusion proteins comprising at least oneNC-1 domain of collagen 18, at least one endostatin domain of collagen18 or a(n) N-terminal peptide(s) of the collagen 18 endostatin domainfor use in diagnosing, preventing, treating or ameliorating fibrosis orfibrosis-associated diseases, a vascular endothelial growth factor(VEGF)-related disease or a matrix metalloproteinase-related disease.

The definitions and embodiments provided as regards the medical uses ofthe protein oligomer or peptide oligomer apply mutatis mutandis to thetherapeutic or diagnostic application and uses of the fusion protein ofthe invention.

Suitable and preferred protein oligomers, fusion proteins, NC-1 domainsof collagen 18, endostatin domains of collagen 18 and N-terminalpeptide(s) of the collagen 18 endostatin domain are defined elsewhereherein and shown in the Figures and the examples. Preferably, saidfusion proteins comprise SEQ ID NO: 3, 4, 7, 9, 10, 13, 15, 18, 19, 20,22, 27 or 29, or N-terminal peptide(s) of the collagen 18 endostatindomain described in Tjin et al., loc. cit., or in U.S. Pat. No.7,524,811. Preferred Fc sequences are depicted in SEQ ID NO. 6, 24, 25,26, 28 or 30.

It is required that the fusion proteins of the invention are able todimerize or oligomerize via an oligomerization domain as definedelsewhere herein. As set forth elsewhere herein, this dimerization oroligomerization is a prerequisite for the binding of the fusion proteinto Fibronectin, VEGF, integrins, MMP-2 and MMP-9, and possibly furtherbinding partners not yet unraveled so far. Preferably, theoligomerization domain is an Fc domain (preferably from or derived fromIgG, more preferably human IgG, even more preferably human IgG1) and/oran artificial oligomerization domain, as specified elsewhere herein.Furthermore, the fusion protein has preferably anti-fibrotic activity,anti-inflammatory, anti-invasive/metastatic, reducing vascularpermeability, anti-angiogenic activity and/or anti-tumorigenic activity.It is also preferred that the fusion protein comprises one or more RGDmotifs and/or PHSRN motifs of Fibronectin, as defined in thisspecification. It is further preferred that the fusion protein is human.

The invention further describes polynucleotides encoding the NC-1monomer, endostatin domain of collagen 18 or N-terminal peptide(s) ofthe collagen 18 endostatin domain and pertains to polynucleotidesencoding the fusion proteins of the invention.

The term “polynucleotide” or “nucleic acid” as used herein refers tosingle- or double-stranded DNA molecules as well as to RNA molecules.Encompassed by the said term is genomic DNA, cDNA, hnRNA, mRNA as wellas all naturally occurring or artificially modified derivatives of suchmolecular species. The polynucleotide may be in an aspect a linear orcircular molecule. Moreover, in addition to the nucleic acid sequencesencoding the NC-1 monomer, endostatin domain of collagen 18 orN-terminal peptide(s) of the collagen 18 endostatin domain or a fusionprotein comprising said monomer or peptide, a polynucleotide maycomprise additional sequences required for proper transcription and/ortranslation such as 5′- or 3′-UTR sequences. In light of the degeneracyof the genetic code, optimized codons may be used in the nucleic acidsequences encoding the NC-1 monomer, endostatin domain of collagen 18 orN-terminal peptides of the collagen 18 endostatin domain or a fusionprotein comprising said monomer or peptide. Thereby, optimal expressionin, e.g., a host cell can be achieved.

It will be understood that the present invention also encompassesvariants of such specific amino acid sequences of the NC-1 monomer,endostatin domain of collagen 18 or N-terminal peptides of the collagen18 endostatin domain or nucleic acid sequences encoding them as long asthese variant sequences also allow for the formation of a protein orpeptide oligomer. Said variants have preferably anti-fibrotic activity,and can have also anti-angiogenic and/or anti-tumor activity as definedelsewhere herein. In an aspect, a sequence variant as used hereindiffers from the specific amino acid sequence or a specific nucleic acidsequence as specified before by one or more amino acid or nucleotidesubstitutions, additions and/or deletions. In another aspect, the saidvariant sequence is at least 50%, at least 60%, at least 70%, at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98% or at least 99% identical to the specific nucleic acid sequence oramino acid sequence of the NC-1 monomer, endostatin domain of collagen18 or N-terminal peptide(s) of the collagen 18 endostatin domain overthe entire length or over at least a stretch of half of the length ofthe specific sequence. Preferably, the said variant sequence is at least50%, at least 60%, at least 70%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identicalto the specific amino acid sequence of the human NC-1 monomer or domainas shown in SEQ ID NO: 4 or the mouse NC-1 monomer or domain as shown inSEQ ID NO: 3, over the entire length. It is also preferred that the saidvariant sequence is at least 50%, at least 60%, at least 70%, at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98% or at least 99% identical to the sequences depicted in SEQ ID NO: 7,9, 10, 13, 15, 18, 19, 20, 22, 27 or 29 over the entire length. The term“identical” as used herein refers to sequence identity characterized bydetermining the number of identical amino acids between sequenceswherein the sequences are aligned so that the highest order match isobtained. It can be calculated using published techniques or methodscodified in computer programs such as, for example, BLASTP or FASTA(Altschul 1990, JMol Biol 215, 403). The percent identity values are, inone aspect, calculated over the entire amino acid sequence or over asequence stretch of at least 50% of the query sequence. A series ofprograms based on a variety of algorithms is available to the skilledworker for comparing different sequences. In this context, thealgorithms of Needleman and Wunsch or Smith and Waterman giveparticularly reliable results. To carry out the sequence alignments, theprogram PileUp (Higgins 1989, CABIOS 5, 151) or the programs Gap andBestFit (Needleman 1970, JMol Biol 48; 443; Smith 1981, Adv Appl Math 2,482), which are part of the GCG software packet (Genetics Computer Group1991, 575 Science Drive, Madison, Wis., USA 53711), may be used. Thesequence identity values recited above in percent (%) are to bedetermined, in another aspect of the invention, using the program GAPover the entire sequence region with the following settings: Gap Weight:50, Length Weight: 3, Average Match: 10.000 and Average Mismatch: 0.000,which, unless otherwise specified, shall always be used as standardsettings for sequence alignments.

The invention further describes a vector comprising the polynucleotideencoding the NC-1 monomer, endostatin domain of collagen 18 orN-terminal peptide(s) of the collagen 18 endostatin domain or a fusionprotein comprising said monomer, domain or peptide.

Preferably, the vector is an expression vector.

The term “vector” encompasses preferably phage, plasmid, viral orretroviral vectors as well as artificial chromosomes, such as bacterialor yeast artificial chromosomes. Moreover, the term also relates totargeting constructs which allow for random or site-directed integrationof the targeting construct into genomic DNA. Such target constructs, inan aspect, comprise DNA of sufficient length for either homologous orheterologous recombination as described in detail below. The vectorencompassing the mentioned polynucleotide, in an aspect, furthercomprises selectable markers for propagation and/or selection in a hostcell. The vector may be incorporated into a host cell by varioustechniques well known in the art. For example, a plasmid vector can beintroduced in a precipitate such as a calcium phosphate precipitate orrubidium chloride precipitate, or in a complex with a charged lipid orin carbon-based clusters, such as fullerens. Alternatively, a plasmidvector may be introduced by heat shock or electroporation techniques.Should the vector be a virus, it may be packaged in vitro using anappropriate packaging cell line prior to application to host cells.Retroviral vectors may be replication competent or replicationdefective. In the latter case, viral propagation generally will occuronly in complementing host/cells.

Moreover, in an aspect, the above-indicated polynucleotide isoperatively linked to expression control sequences allowing expressionin prokaryotic or eukaryotic host cells or isolated fractions thereof inthe said vector. Thus, in an aspect, the vector is an expression vector.Expression of the polynucleotide comprises transcription of thepolynucleotide into a translatable mRNA. Regulatory elements ensuringexpression in host cells are well known in the art. In an aspect, theycomprise regulatory sequences ensuring initiation of transcriptionand/or poly-A signals ensuring termination of transcription andstabilization of the transcript. Additional regulatory elements mayinclude transcriptional as well as translational enhancers. Possibleregulatory elements permitting expression in prokaryotic host cellscomprise, e.g., the lac-, trp- or tac- promoter in E. coli, and examplesfor regulatory elements permitting expression in eukaryotic host cellsare the AOX1- or the GAL1- promoter in yeast or the CMV-, SV40-,RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or aglobin intron in mammalian and other animal cells. Moreover, inducibleexpression control sequences may be used in an expression vector. Suchinducible vectors may comprise tet or lac operator sequences orsequences inducible by heat shock or other environmental factors.Suitable expression control sequences are well known in the art. Besideelements which are responsible for the initiation of transcription suchregulatory elements may also comprise transcription termination signals,such as the SV40-poly-A site or the tk-poly-A site, downstream of thepolynucleotide. In this context, suitable expression vectors are knownin the art such as Okayama-Berg cDNA expression vector pcDV1(Pharmacia), pBluescript (Stratagene), pCDM8, pRc/CMV, pcDNA1, pcDNA3(Invitrogen) or pSPORT1 (Invitrogen). Preferably, said vector is anexpression vector and a gene transfer or targeting vector. Expressionvectors derived from viruses such as retroviruses, vaccinia virus,adeno-associated virus, herpes viruses, or bovine papilloma virus, maybe used for delivery of the polynucleotide or vector into a targetedcell population. Methods which are well known to those skilled in theart can be used to construct recombinant viral vectors; see, forexample, the techniques described in Sambrook, Molecular Cloning ALaboratory Manual, Cold Spring Harbor Laboratory (2001) N.Y. andAusubel, Current Protocols in Molecular Biology, Green PublishingAssociates and Wiley Interscience, N.Y. (1994).

The invention further describes a host cell comprising thepolynucleotide encoding the NC-1 monomer, endostatin domain of collagen18 or N-terminal peptide(s) of the collagen 18 endostatin domain or afusion protein comprising said monomer or peptide, or the vectorcontaining such polynucleotide.

The term “host cell” as used herein as used herein encompassesprokaryotic and eukaryotic host cells. In an aspect the host cell is abacterial cell. In one aspect, the said bacterial host cell is an E.colihost cell. Such a bacterial host cell may be used, e.g., forreproduction of the mentioned polynucleotide or vector.

A eukaryotic host cell, in an aspect, is a cell which comprises thepolynucleotide encoding the NC-1 monomer, endostatin domain of collagen18 or N-terminal peptide(s) of the collagen 18 endostatin domain, or afusion protein comprising said monomer, domain or peptide, or the vectorwherein said polynucleotide or vector are expressed in the host cell inorder to generate the protein or peptide or oligomer thereof. Thepolynucleotide may be introduced into a host cell either transiently orstably. In an aspect, the eukaryotic host cell may be a cell of aeukaryotic host cell line which stably expresses the polynucleotide. Inanother aspect, the host cell is a eukaryotic host cell which has beentransiently transfected with the polynucleotide or vector and whichexpresses the polynucleotide. In another aspect, the said cell is a cellwhich has been genetically engineered to produce the protein or peptide.How such cells can be genetically engineered by molecular biologytechniques is well known to the skilled person.

The present invention also relates to a kit comprising the proteinoligomer or fusion protein of the invention.

The term “kit” as used herein refers to a collection of the proteinoligomer or fusion protein of the present invention which may or may notbe packaged together. It is required that the fusion protein is able tooligomerize, as explained elsewhere herein. The kit can encompassfurther components for formulating the protein oligomer or fusionprotein of the present invention as a pharmaceutical or diagnosticcomposition. The components of the kit may be comprised by separatevials (i.e. as a kit of separate parts) or provided in a single vial.Moreover, it is to be understood that the kit of the present inventionis to be used for the therapy or diagnosis of the diseases referred toherein above. In one aspect, it is envisaged that all components areprovided in a ready-to-use manner for practicing the therapeutic ordiagnostic uses referred to herein. In a further aspect, the kitcontains instructions for carrying out the said uses. The instructionscan be provided by a user manual in paper- or electronic form.

The present invention further pertains to the use of the proteinoligomer or fusion protein of the invention for generating and,preferably, improving a pharmaceutical composition mimicking NC1.

In addition, the present invention relates to the use of the proteinoligomer or fusion protein of the invention for the development ofNC1-mimetics or mimetics of oligomeric endostatin for the treatment ordiagnosis of fibrosis-related diseases, VEGF-related diseases, orMMP-related diseases, as defined herein.

Finally, the present invention relates to the use of the proteinoligomer or fusion protein of the invention for the development ofNC1-mimetics or mimetics of oligomeric endostatin for the modulation offibronectin function.

What is to be understood by mimetics has been explained elsewhereherein.

Fibronectin (FN) is a pleiotropic molecule with a number of activitiesand binding partners in matrix remodeling, immune response, invasion,and epithelial-to-mesenchymal transition. The more it is understood howthe oligomer of the invention interacts with Fibronectin function, themore specific those functions can be targeted. For example, if theprotein oligomer of the invention binds to the same heparin binding siteof FN (heparin binding site II) as also VEGF does, it can be analyzedwhat is the implication if FN is being trapped for VEGF binding or whatis the effect for binding to integrins or MMPs. FN does a number ofeffects that are differentially modulated by NC1. The more is knownabout these modulations, the better they can be targeted the way natureintended via NC-1.

Particularly preferred protein oligomers, peptide oligomers and fusionproteins for the medical and diagnostic uses of the invention aredepicted in the Figures and examples.

SEQUENCES

The sequences show:

-   -   SEQ ID NO: 1: murine Collagen 18    -   SEQ ID NO: 2: human Collagen 18    -   SEQ ID NO: 3: NC-1 domain of murine Collagen 18    -   SEQ ID NO: 4: NC-1 domain of human Collagen 18    -   SEQ ID NO: 5: murine Fc domain    -   SEQ ID NO: 6: human Fc domain    -   SEQ ID NO: 7: murine Superstatin    -   SEQ ID NO: 8: murine Fibronectin motif    -   SEQ ID NO: 9: murine N-terminal zinc-binding domain Endostatin    -   SEQ ID NO: 10: human N-terminal zinc-binding domain Endostatin    -   SEQ ID NO: 11: murine RGD motif    -   SEQ ID NO: 12: human RGD motif    -   SEQ ID NO: 13: human Superstatin    -   SEQ ID NO: 14: human Superstatin with His at positions 1 and 3        replaced by Ala    -   SEQ ID NO: 15: human Superstatin with Gln at position 7 replaced        by Cys    -   SEQ ID NO: 16: human Superstatin with “RGD” motif replaced by        “RAD” motif    -   SEQ ID NO: 17: murine integrin-binding motifs of Fibronectin    -   SEQ ID NO: 18: mEndostatin (murine)    -   SEQ ID NO: 19: hEndostatin (human)    -   SEQ ID NO: 20: mP1 peptide (murine; N-terminal)    -   SEQ ID NO: 21: E4 peptide of Endostatin (human; C-terminal)    -   SEQ ID NO: 22: hP1 peptide of Endostatin (human; N-terminal)    -   SEQ ID NO: 23: Enterokinase cleavage site    -   SEQ ID NO: 24: Fc sequence of wildtype human IgG1    -   SEQ ID NO: 25: Fc sequence “knob” human IgG1 Fc: S354C/T366W    -   SEQ ID NO: 26: Fc sequence “hole” human IgG1 Fc        Y349C/T366S/L368A/Y407V    -   SEQ ID NO: 27: NC-1-enterokinase site-linker-human IgG1 Fc with        “knob” mutations (S354C/T366W)    -   SEQ ID NO: 28: Human IgG1 Fc with “hole” mutations        Y349C/T366S/L368A/Y407V    -   SEQ ID NO: 29: Human IgG1 Fc with “knob” mutations        (S354C/T366W)-linker-enterokinase site-NC-1    -   SEQ ID NO: 30: Human IgG1 Fc with “hole” mutations        Y349C/T366S/L368A/Y407V

SEQ ID NOs: 1-17 are also depicted in WO 2013/026913, the disclosurecontent of which is incorporated herewith by reference. The amino acidsequence of human Fibronectin is shown in UniProt accession numberP02751. The amino acid sequence of the human matrix metalloproteinase-2(MMP-2) is shown in UniProt accession number P08253. The amino acidsequence of the human matrix metalloproteinase-9 (MMP-9) is shown inUniProt accession number P14780. The amino acid sequence of humanvascular endothelial growth factor (VEGF-A) is shown in UniProtaccession number P15692. The recognition and cleavage site ofenterokinase and appropriate linker regions are described, e.g., in Loet al., 1998, Protein Engineering 11(6), 1998, p. 495-500.

FIGURES

The Figures show:

FIG. 1 . The topology of collagen XVIII and Fc-endostatin as well asphysiological size of endostatin molecules in different tissues. (A) Thestructures of collagen XVIII consists of a signal peptide, frizzleddomain, triple helical repeats, NC1 domain. The ES motif isphysiologically trimerized in the context of NC1. (B) Schematic ofsynthesis of two ES domain to IgG Fc domain. (C,D) The contents ofendostatin were found in mouse brain, skeletal muscle, heart, kidney,testis and liver tissue extracts and serum by immunoblot. Imagesreproduced from Kuo, C. J., et al., Oligomerization-dependent regulationof motility and morphogenesis by the collagen XVIII NC1/endostatindomain. J Cell Biol, 2001. 152(6): p. 1233-46.; Sasaki, T., et al.,Structure, function and tissue forms of the C-terminal globular domainof collagen XVIII containing the angiogenesis inhibitor endostatin. EMBOJ, 1998. 17(15): p. 4249-56.]

FIG. 2 . The sequences of endostatin, N-terminal peptides (mP1) and(hP1) and C-terminal E4 (CE4) peptide.

FIG. 3 . Reduced radiation induced lung fibrosis development afterFc-Endo and mP1-treatment. (A) Micro-CT imaging of control and treatmentgroups at the end point of 24 weeks post irradiation. Massive fibrosiswas found in mice that received sham photon irradiation (X-20Gy), withlimited effective ventilation space left in the lung. Interstitialfibrosis was also significant in irradiated lung after CE4 treatment(CE4+X). Much clear lung parenchyma was found in irradiated lungstreated with mP1-Endo (mP1+X) and particularly Fc-Endo-treated mice(Fc-Endo+X). (B) Quantitative clinical CT measurement found asignificantly reduced mean lung density (MLD) as well as total lungvolume (LV) loss in Fc-Endo+X and mP1-Endo+X groups, respectively. (C)The fibrosis indices (FIs) of Fc-Endo+X and mP1-Endo+X treatment groupswere significantly decreased compared to that of the IR-only group(X20Gy). There was no statistical difference between the CE4-Endo+Xtreatment and IR-only groups. (D) Great survival benefits were found forthe Fc-Endo+X and mP1-Endo+X groups but not for the CE4+X treated group(*P<0.05, **P<0.01, ***P<0.001, ns=not statistically significant).

FIG. 4 . Improved clinical parameter and pathohistological presentationafter Fc-Endo and mP1-Endo treatment in irradiated lungs. (A, B) Bloodgas analysis demonstrated an increased partial pressure of carbondioxide (pCO₂) in the blood and decreased level of pH (acidosis) inIR-only (X-20Gy) group at the end point of 24 weeks after irradiation.PCO₂ and pH levels of mice treated with Fc-Endo+X or mP1-Endo+X weresignificantly ameliorated. (C) All treatment groups containingendostatin polypeptide fragments benefited in terms of weight gain.Among those, Fc-Endo was found to have the most significant improvement.(D) Histopathological examination confirmed significantly reducedinflammation and fibrosis in Fc-Endo+X and mP1-Endo+X treatment groups.(E) No histopathological difference was found among non-irradiatedFc-Endo, mP1-Endo and CE4-Endo treatment groups (*P<0.05, **P<0.01,***P<0.001).

FIG. 5 . M2 polarization, gene- and protein expression after Fc-Endo+Xtreatment. (A) The transcription signatures of ECM proteins weresuppressed by Fc-Endo+X treatment. (B) Fc-Endo was also found toattenuate radiation induced pro-fibrotic M2 macrophage polarization. (C)Fc-Endo treatment reversed radiation induced FGF2- and loss of CD31expression. In contrast, the expression of anti-fibrosis HGF wasincreased by Fc-Endo+X vs. X20Gy treatment. (*P<0.05, **P<0.01,***P<0.001).

FIG. 6 . Fc-Endo reduced fibrosis after high-LET carbon irradiation. (A)Micro-CT imaging at the end point of 24 weeks post irradiation. Massivefibrosis was found in mice irradiated with carbon-ion 12.5 Gy (C12.5),with limited effective ventilation space left in the lung. However, thelung architecture was well preserved in mice treated with Fc-Endo+C12.5.(B) Quantitative clinical CT measurement showed a significantly reducedmean lung density (MLD) as well as total lung volume (LV) loss in theFc-Endo+C12.5- vs. C12.5-group. (C) The fibrosis indices (FIs) ofFc-Endo+C12.5 treatment group were significantly lower than the FIs ofthe C12.5 alone group. (D) Pathohistological examination confirmedsignificantly reduced inflammation and fibrosis after Fc-Endo+C12.5 vs.carbon irradiation alone (*P<0.05, **P<0.01, ***P<0.001).

FIG. 7 . Related evidence for inhibition of fibrosis by Fc-Endotreatment after carbon-ion irradiation. (A) Fc-Endo was also found toattenuate pro-fibrosis M2 macrophage infiltration. (B) The expression ofpro-fibrosis FGF2 was decreased by Fc-Endo. (C) The expression ofanti-fibrosis HGF was increased by Fc-Endo. (D) The loss of endothelialmarker CD31 was reversed by Fc-Endo. (*P<0.05, **P<0.01, ***P<0.001).

FIG. 8 . A demonstration of precise mice thorax irradiation by carbonions irradiation (Carbon ions 12.5Gy at the SOBP). (A) Mouse was fixedin a specially designed holder for thoracic irradiation. (B) Theparticle dosing in lung was homogenous as verified by the entrance andexit films (Kodak EDR2). (C) PET/CT beam verification immediately afterirradiation confirmed a precise dose deposition in the lung area. Thebreath motion has been also taken into account.

FIG. 9 . Fibronectin binding of oligomeric endostatin. Exclusive bindingof NC1 and endostatin (ES) dimer but not monomer to fibronectin (FN).Elisa plate was coated with human fibronectin. After blocking with BSA,endostatin monomer, dimer and NC1 were used as ligands at concentrationsindicated. For detection of endostatin bound to fibronectin,anti-endostatin antibodies were employed.

FIG. 10 . VEGF binding of oligomeric endostatin. Exclusive binding ofNC1 and endostatin (ES) dimer but not endostatin monomer to the vascularendothelial growth factor (VEGF). Elisa plate was coated with humanVEGF. After blocking with BSA, endostatin monomer, dimer and NC1 wereused as ligands at concentrations indicated. For detection of endostatinbound to VEGF, anti-endostatin antibodies were employed.

FIG. 11 . MMP-2/9 binding of oligomeric endostatin. Binding of NC1,endostatin (ES) dimer and Fc-Endostatin (dimerization over Fc moiety) toMMP-2 (A) and MMP-9 (B), respectively. In contrast, endostatin monomershows a weak binding to the MMPs. Elisa plate was coated with humanMMP-2 or MMP-9, respectively. After blocking with BSA, endostatin (ES)monomer, endostatin (ES) dimer, Fc-endostatin (Fc-ES), Fc-control (Fc)and NC1 were used as ligands at concentrations indicated. For detectionof endostatin bound to MMPs, anti-endostatin antibodies were employed.

FIG. 12 . Loss of dimeric endostatin binding to MMP-2 and fibronectinafter enterokinase digestion of Fc-endostatin (humanFcE) into endostatinmonomer and Fc-dimer. (A) SDS-polyacrylamide electrophoresis of hFcEdigestion by enterokinase. Lane 1 (Protein Markers). Lane 2: hFcE. Lane3; upper band is Fc-dimer and lower band is endostatin monomer followingdigestion with enterokinase. Samples in 2 and 3 are under non-reducedconditions. Lane 5: hFcE. Lane 6: upper band Fc and lower bandendostatin monomer following digestion. Lanes 5&6 were performed inreduced conditions. (B) Elisa assay as described above detects bindingof dimeric endostatin (FcE) to fibronectin and MMP-2, respectively.Note, enterokinase digestion leading to monomeric endostatin and dimericFc resulting in significantly reduced binding to both candidate targetmolecules.

FIG. 13 . Schematic illustration of protein oligomers for use in theinvention. (A) Protein oligomer comprising N-terminal homodimeric Fcfusion constructs. Wildtype human IgG1 Fc is fused to the N-terminus ofNC-1 or endostatin via a protease-cleavable linker. (B) Protein oligomercomprising C-terminal homodimeric Fc fusion constructs. Wildtype humanIgG1 Fc is fused to the C-terminus of NC-1 or endostatin via aprotease-cleavable linker.

FIG. 14 . “Knobs-into-holes” (KiH)-engineered NC-1-Fc or endostatin-Fcfusion constructs (A) Heterodimer comprising a human IgG1 Fc with “knob”mutations fused to the N-terminus of the NC-1 or endostatin via aprotease-cleavable linker, and a human IgG1 Fc with “hole” mutations.(B) Heterodimer comprising a human IgG1 Fc with “knob” mutations fusedto the C-terminus of the NC-1 or endostatin via a protease-cleavablelinker, and a human IgG1 Fc with “hole” mutations.

The invention will now be illustrated by examples which shall, however,not be construed as limiting the scope of the invention.

EXAMPLES Example 1: Endostatin

Fc-endostatin (Fc-Endo) is a fusion protein of endostatin to the Fcregion of a human IgG. It shows significantly improved pharmacokineticsand biologic efficacy relative to endostatin [Lee, T. Y., et al.,Linking antibody Fc domain to endostatin significantly improvesendostatin half-life and efficacy. Clin Cancer Res, 2008. 14(5): p.1487-93]. Moreover, fusion of two endostatin monomeric molecules to IgGFc domain in Fc-endostatin leads to a synthetic dimerization of themolecule (FIG. 1B). Interestingly, oligomerization of endostatin waspreviously shown to confer additional properties to the molecule[Sudhakar, A., et al., Human tumstatin and human endostatin exhibitdistinct antiangiogenic activities mediated by alpha v beta 3 and alpha5 beta 1 integrins. Proc Natl Acad Sci U S A, 2003. 100(8): p. 4766-71].Of note, the natural non-collagenous region of collagen 18 (NC-1)consists of three endostatin monomers connected over a proteasesensitive hinge- and trimerization regions, respectively (FIG. 1A).Hence, the monomeric endostatin could be considered the finalproteolytic fragment of the original trimeric molecule. Indeed, largerfragments than the monomeric endostatin were physiologically found indifferent tissues and serum (FIG. 1C). In the same context, the presentinventors have shown that oligomerization is a prerequisite for bindingof Fc-endostatin and NC-1 to fibronectin (FN), whereas the endostatinmonomer does not bind FN (WO 2013/026913). FN is recognized as a keyplayer in the pathogenesis of fibrosis, leading to a wide spectrum ofdownstream and associated pro-fibrotic signaling cascades. For examples,FN is reported to bind integrin alpha 5 (ITGA5B) and αvβ₃ [Torres, P.H., G. L. Sousa, and P. G. Pascutti, Structural analysis of theN-terminal fragment of the antiangiogenic protein endostatin: amolecular dynamics study. Proteins, 2011. 79(9): p. 2684-92], which areassociated with fibrosis promotion. Hence, oligomerization of endostatine.g. via Fc-endostatin may have implications for its pleiotropicfunctions.

Yamaguchi et al. reported that endostatin via its C-terminal domain (E4peptide) has elicited anti-fibrosis effects [Yamaguchi, Y., et al., Apeptide derived from endostatin ameliorates organ fibrosis. Sci TranslMed, 2012. 4(136): p. 136ra71]. However, the zinc binding domain hasbeen previously confined to the N terminus (endostatin mP1 peptide) andwas critical to numerous functions of the molecule [Tjin, R. M., et al.,A 27-amino-acid synthetic peptide corresponding to the NH2-terminalzinc-binding domain of endostatin is responsible for its antitumoractivity. Cancer Research, 2005. 65(9): p. 3656-3663]. In the followingexamples, the present inventors aim to better understand the impact ofoligomerization (Fc-endostatin) as well as N- vs. C-terminal fragmentsof endostatin (N-terminal endostatin peptide mP1, SEQ ID NO: 20;C-terminal endostatin peptide E4 or CE4, SEQ ID NO: 21) on modulatingradiation-induced lung fibrosis.

Example 2: Endostatin Administration

Mice were treated with mFc-endostatin (murine endostatin as depicted inSEQ ID NO: 18 fused to the Fc fragment of the murine immunoglobulin γ-2achain, as described in Bergers et al., Science 284(5415), p. 808-812,1999) from 3 days prior to irradiation till the end of the trials, at adose of 100 μg/mouse every 5 days delivered subcutaneously. In parallel,endostatin peptides groups were administrated either mP1 endostatin(N-terminus; SEQ ID NO: 20) or E4 (or CE4) peptide (C-terminus; SEQ IDNO: 21; see Yamaguchi, Y., et al., A peptide derived from endostatinameliorates organ fibrosis. Sci Transl Med, 2012. 4(136): p. 136ra71),at a dose of 100 μg/mouse/b.i., subcutaneously, in addition toirradiations. Control groups were treated with PBS, mFc-endostatin, mP1endostatin or E4 peptide alone and received no irradiation.

Example 3: Photon Irradiation and High-LET Carbon Irradiation

Total thoracic irradiation was performed as described previously withmodifications (Abdollahi, J. Exp. Med. 2005,http://www.ncbi.nlm.nih.gov/pubmed/15781583).

Whole thoracic irradiation was administrated to 8-week-old C57BL/6 mice(Taconis, Bomholtvej, Denmark). Mice were maintained under specificpathogen-free conditions, and experiments were performed in compliancewith institutional guidelines as approved by the Animal Care and UseCommittee of the German Cancer Research Center (DKFZ). Prior to thoracicirradiation, mice were anesthetized by an intraperitoneal application of0.36 ml/kg Rompun 2% (Bayer HealthCare) and 0.54 ml/kg ketamine 10%(Pfizer).

Particle irradiations were performed at the Heidelberg Ion-Beam TherapyCenter (HIT), Heidelberg, Germany. The irradiation setup is shown below(FIG. 8 ). Carbon ions (12C) were applied at the spread-out Bragg peak(SOBP, 252.400-270.550 MeV/u) with linear energy transfer (LET)=70-157keV/μm (mean at 86 keV/μm).

Photon irradiation was done with an Artiste linear accelerator (Siemens,Germany) with 6 MV and at a dose rate of 3 Gy/min at DKFZ, Heidelberg,Germany.

All irradiation plans were adjusted by anatomical and radiologicalestimation, ensuring full coverage of the lung area and sparingneighboring tissue at the maximum. PET/CT (Biograph mCT, Siemens)imaging was applied also for beam verification immediately aftercarbon-ion irradiation.

Example 4: Fibrosis Index (FI) Calculation

A clinical PET/CT scanner (Biograph mCT, Siemens) was applied forquantitative CT imaging pre-irradiation (pre-IR) and every 4 weekspost-irradiation (post-IR). CT imaging was performed under isofluraceanesthesia (2% isoflurane, 2 l/min oxygen). The standard protocol forthe CT portion was as follows: 80 kV with 80 mA, a pitch of 0.6 mm,slice thickness of 0.6 mm and acquisition time of 32 s. Images werereconstructed using the filter kernel H50s (Siemens) into a transaxialfield of view of 138×138 mm2 as a 512×512 matrix, where three animalswere included in one scan. X-ray exposure was approximately 4 mGy perscan in the total field of view, roughly less than 1 mGy per animal.

Images acquired from the clinical CT scanner were viewed and analyzed inOsiriX Imaging Software (OsiriX v.3.9.4 64-bit version, Pixmeo SARL,Switzerland) and MITK software (Medical and Biological Informatics,German Cancer Research Center). Because of the relatively low resolutionof the images, the HU intensities of microvasculature were averaged withthe surrounding air-contained tissues. The lung, together with all themicrostructures, was thereby segmented using a three- dimensional (3D)regional growing algorithm with a lower threshold of −900 HU and anupper threshold of −100 HU. A lower threshold of −600 HU was used onanimals with emphysema (−450 HU). Trachea and primary bronchi weremanually resected upon segmentation. After mean HU value and volume sizewere calculated within the segmented area, a histogram of the same lungregion binned in an interval of 10 HU was extracted in order to achievea more reliable evaluation that was insensitive to the selection ofthreshold values. Micro-CT imaging was performed using both the micro-CTcomponent of a prototype SPECT-CT-OT system and Inveon SPECT/PET/CT(Siemens, Germany) at the corresponding time points (pre-IR and every 4weeks post-IR) for further validation of clinical CT results. Forprototype SPECT-CT-OT system, CT acquisitions were performed at 40 kVtube voltage, 0.4 mA anode current, 1 second acquisition time perprojection, 240 projections per 360-degree rotation. Images werereconstructed into a matrix of 512×512×1024 with an isotropic voxel sizeof 0.065 mm. For Inveon SPECT/PET/CT, CT acquisition were applied as 80kV tube voltage, 0.5 mA anode current, 1 second acquisition time perprojection, 720 projections per 360-degree rotation, with an effectivepixel size of 19.29 μm. The micro-CT data were viewed and analyzed withMITK software. Segmentation of the lung area was performed manually for10 successive transaxial CT slices. HU values for each voxel in theselected volumes of interest were exported to calculate the mean HUvalue, and, afterwards, used to generate a histogram.

Lung density, represented by the mean HU value of the entire lung areain CT, was calculated from segmented lungs. Fibrosis index (FI) wasproposed based on CT measurement of mean lung density (MLD) inHounsfield unit (HU) and lung volume as:

${{Fibrosis}{index}( {FI} )} = \sqrt{\Delta\overset{\_}{ {HU}\uparrow } \times \Delta\overset{\_}{ V\downarrow }}$

where ΔHU is an average of increased HU value from segmented lungs; isthe decreased lung volume with reference to age-matched control.

Example 5: Attenuation of Photon Irradiation-Induced Lung Fibrosis byEndostatin Fc-Endostatin and mP1 Endostatin Inhibited Lung Fibrosis andProlonged Survival

Mice were treated with Fc-endostatin (Fc-Endo), N-terminus endostatinpeptide (mP1) or C-terminus endostatin E4 peptide (CE4) (see FIG. 2 )combined with photon 20 Gy whole thoracic irradiation (see Example 3).Micro-CT imaging revealed diffuse ground glass opacities andarchitectural distortion in 20 Gy-irradiated tissue, indicating amassive interstitial fibrosis generation. Severe fibrotic lungparenchyma was seen also in the CE4 combined irradiation group. Incontrast, remarkably reduced fibrosis were observed in Fc-Endo and mP1treatment groups (FIG. 3 ).

Quantitative clinical CT follow-up was completed at the end point of 24weeks after irradiation (FIG. 3B). Compared to the ionizing radiation(IR)-only condition, significantly reduced mean lung density (MLD) wasfound in the Fc-Endo+X and mP1+X treatment groups (P<0.001, P<0.05,respectively). In line with this, total lung volume (LV) was preservedin those groups, whereas significant loss of LV occurred in IR-only mice(P<0.001, P<0.05, respectively, for Fc-Endo+X and mP1+X relative toIR-only). No significant difference was observed in the CE4+X treatedgroup in terms of MLD or decreased LV compared to IR-only mice (P>0.05).No statistical difference was found in Fc-Endo, mP 1 or CE4 treatedgroups, in comparison to control groups in the same parameters (MLD andLV, data not shown).

Based on the inventors' radiation induced lung fibrosis (RILF), thefibrosis index (FI) was considered the most reliable and robustindicator for quantitative assessment of lung fibrosis (see Example 4).Remarkably attenuated FI levels were observed in the Fc-Endo+X (around3.03, 43% decrease) and mP1+X (around 1.86, 26% decrease) treatmentgroups (P<0.001, P<0.05, respectively). However, there was nostatistically significant difference between the CE4+X-administered andIR-only groups (P=0.43) (FIG. 3C).

The reduced radiation induced FI values correlated with a survivalbenefit in the Fc-Endo and mP1 treatment groups; e.g., the survival rateat the end of the observation period (25 weeks post IR) was 80% forFc-Endo+X and 60% for mP1+X arm, respectively, versus only 10% inIR-only mice (P<0.01 and P<0.05, respectively). The inventors did notobserve a statistically significant difference between CE4+X and IR-onlygroups (FIG. 3D).

Example 6: Fc-Endostatin and mP1 Endostatin Improved Pulmonary Function

As an accompanying symptom of lung fibrosis, the deterioration inpulmonary function was studied in all groups (FIG. 4A, B). The IR-onlygroup had significantly higher pCO₂ and lower pH compared to the controlgroup (P<0.05, P<0.001, respectively), indicating a chronic respiratoryacidosis because of serious impairment in ventilation. In contrast,these measurements did not differ significantly between Fc-Endo- ormP1+X treated groups and the control group (P>0.05, P>0.05,respectively), revealing a favorable respiratory function in thosegroups.

In comparison to the IR-only group (X 20 Gy), the most well-protectedlung function in terms of pCO₂ and pH was achieved with Fc-Endo+Xtreatment (P<0.05, P<0.001, respectively). The next best outcome wasprovided by mP1+X, for which pCO₂ and pH were also significantlydifferent from IR-only (X20Gy) conditions (P<0.05, P<0.05,respectively).

No significant benefits (pCO₂ and pH) were found in mice treated withCE4+X compared to IR-only mice (P>0.05, P>0.05, respectively). Weightloss at the end point was evaluated (FIG. 4C). All endostatin treatmentgroups were observed with less weight loss compared to IR-only mice (allP<0.05). In particular, Fc-Endo+X-treated mice had an advantage overmP1-Endo−X and CE4-Endo+X-treated mice in terms of weight (both P<0.05relative to Fc-Endo).

Histopathological analysis suggested clear improvements in inflammatorycell infiltration, septal thickness and alveolar architecture in micethat received Fc-Endo+X or mP1-Endo+X, compared to IR-only mice. TheFc-Endo+X and mP1-Endo+X treatment groups also showed markedly lesscollagen deposition and scarring in trichrome stainings (FIG. 4D). Therewas no difference in non-irradiated lung treated with Fc-Endo, mP1-Endoor CE4-Endo (FIG. 4E).

Example 7: Effects of Fc-Endostatin on M2 Polarization, Gene- andProtein Regulation

Aberrant ECM remodeling is a characteristic feature of pulmonaryfibrosis. The inventors found that a variety of key ECM proteinsincluding tenascin C, collagen I and III, elastin, fibrillin, α-actinand MMPs were suppressed or ‘switched off’ by Fc-Endo+X at thetranscriptional level (FIG. 5A). In concert with this,immunohistochemistry results suggested a reduction of M2 macrophageinflux in Fc-Endostatin+X vs. 20Gy irradiated lung (FIG. 5B).

Radiation induced pulmonary fibrosis (X20Gy) was associated with reducedCD31 and enhanced basic fibroblast growth factor (bFGF or FGF2) proteinlevels. Addition of Fc-Endo to radiotherapy reversed this phenotype tothe levels detected in the sham treated control (FIG. 5 ). Intriguingly,hepatocyte growth factor (HGF), which has been recently associated withanti-fibrotic properties [Crestani, B., et al., Hepatocyte growth factorand lung fibrosis. Proc Am Thorac Soc, 2012. 9(3): p. 158-63; Phin, S.,et al., Imbalance in the pro-hepatocyte growth factor activation systemin bleomycin-induced lung fibrosis in mice. Am J Respir Cell Mol Biol,2010. 42(3): p. 286-93] was expressed at a much higher level afterFc-Endo+X, compared to IR-alone (X-20Gy) (P<0.05) (FIG. 5D).

Example 8: Inhibition of Carbon-Ion-Induced Pulmonary Fibrosis byFc-Endostatin Fc-Endostatin Inhibited Lung Fibrosis Induced by CarbonIons

Given that Fc-endostatin was more effective than other endostatinpeptide fragments at inhibiting photon-induced lung fibrosis, theinventors next studied the efficacy of Fc-endostatin to modulatefibrosis induced by high-LET carbon irradiation.

Micro-CT imaging showed diffuse fibrotic lungs, after carbon-ion 12.5 Gyirradiation (C12.5). In contrast, remarkably reduced fibrosis was seenin the Fc-Endo+C12.5 treatment group (FIG. 6A).

Quantitative clinical CT follow-up at the endpoint of 24 weeks wasperformed (FIG. 6B). Lung density (MLD) in the Fc-Endo+C12.5 group wassignificantly lower than that in the carbon alone (C12.5) group(P<0.01). In line with this, total LV was also preserved inFc-Endo+C12.5-treated mice, whereas there was significant loss in totalLV for carbon alone (C12.5) mice (P<0.01).

FI was considered the most important indicator in lung fibrosisassessment (see Example 4). The FI of the Fc-Endo+C12.5 group wasnotably lower than that of the carbon irradiated (C12.5) group (P<0.001)(FIG. 6C).

Histopathological analysis suggested clear improvements in inflammation,septal thickness and the alveolar architecture for mice receivingFc-Endo+C12.5, compared to carbon irradited (C12.5) mice. Likewise, lesscollagen deposition and scarring was found in trichrome stainings forFc-Endo+C12.5-treated mice (FIG. 6D).

Example 9: Immunological and Molecular Confirmation of FibrosisInhibition after High LET Irradiation by Fc-Endostatin

Mice receiving Fc-endostatin treatment had a clear reduction ofpro-fibrotic M2 macrophages (CD206 and CLL22 positive), in carbonirradiated lungs (FIG. 7A). In line with endostatin effects on photonirradiated lungs, a reversal of FGF2 induction and loss of CD31 proteinlevels was found after Fc-Endo+C12.5 vs. carbon radiation alone (FIG.7B). Moreover, Fc-endostatin treatment resulted in elevatedanti-fibrotic HGF protein levels in carbon irradiated lungs (P<0.05)(FIG. 7C).

Together, these data confirm the relevance of M2 polarization, reducedintact lung architecture consisting of blood gas barrier (CD31 positivemicrovessels) and growth factor/cytokine profile (FGF) in development offibrosis independent of radiation quality. Fc-endostatin efficientlyreversed this phenotype in both carbon- and photon-irradiation models.

Example 10: Possible Mechanisms of Fc-Endostatin in Inhibiting Fibrosis

The inventors found that Fc-endostatin (Fc-Endo) and N-terminusendostatin (mP1 endostatin) peptide were effective inhibitors of lungfibrosis induced by photon or carbon-ion irradiation. Fc-endostatin wassuperior to mP1 in terms of survivals, radiological, physiological,histological examinations, M2 macrophage polarization and Th2-biasedimmunity, ECM composition, cellularity alternations, etc. This could bethe consequence of improved pharmacokinetics of Fc-endostatin withlonger half-life (exposure) as reported for the anti-cancer activity ofthis compound [Lee, T. Y., et al., Linking Antibody Fc Domain toEndostatin Significantly Improves Endostatin Half-life and Efficacy.Clinical Cancer Research, 2008. 14(5): p. 1487-1493]. Alternatively,different mechanism of action might govern the anti-fibrotic effect ofFc-endostatin. Considering that endostatin is a proteolytic fragment ofcollagen 18 non-collagenous domain 1 (NC-1) which is physiologically atrimer, dimerization of endostatin via Fc-conjugation might represent amore physiologic correlate of this endogenous protein. Interestingly,endostatin plasma levels were found to be enhanced in patients withpulmonary fibrosis. The inventors have previously shown thatFc-endostatin as a synthetic dimer can bind to fibronectin (FN), whereasendostatin monomer does not (see WO 2013026913). FN is thought to have acentral role in initiation and perturbation of fibrosis development [To,W. S. and K. S. Midwood, Plasma and cellular fibronectin: distinct andindependent functions during tissue repair. Fibrogenesis Tissue Repair,2011. 4: p. 21]. Therefore, it is tempting to speculate thatFc-endostatin binding to FN, leads to a wide spectrum of downstreamanti-fibrotic signal cascades.

Matrix formation requires FN, integrins and molecule adhesion to thecytoskeleton [Schwarzbauer, J. E. and D. W. DeSimone, Fibronectins,their fibrillogenesis, and in vivo functions. Cold Spring Harb PerspectBiol, 2011. 3(7)]. Integrin-mediated connective tissue production is theessential pathway in fibrogenesis. Most integrins bind to FN through theRGD loop in FNIII₁₀ and the neighboring PHSRN sequence in FNIII₉. It iswell accepted that the binding of pro-fibrotic integrins (e.g., α₅β₁,αvβ₁, αvβ₃) to FN is a key step in the progression of FN-matrix assembly[Takahashi, S., et al., The RGD motif in fibronectin is essential fordevelopment but dispensable for fibril assembly. J Cell Biol, 2007.178(1): p. 167-78; Leiss, M., et al., The role of integrin binding sitesin fibronectin matrix assembly in vivo. Curr Opin Cell Biol, 2008.20(5): p. 502-7].

The present inventors found a strong activation of aIIb integrin byFc-endostatin. In particular, Kindlin-3, the key molecule to activatingintegrin aIIb was also found to be highly up-regulated transcriptionally(data not shown). Integrin aIIb binds to FN at the FNIII₉₋₁₀, which arethe same sites for those pro-fibrotic integrins [Leiss, M., et al., Therole of integrin binding sites in fibronectin matrix assembly in vivo.Curr Opin Cell Biol, 2008. 20(5): p. 502-7., Chada, D., T. Mather, andM. U. Nollert, The synergy site of fibronectin is required for stronginteraction with the platelet integrin alphaIIbbeta3. Ann Biomed Eng,2006. 34(10): p. 1542-52]. Hence, in addition to FN binding, endostatininduced integrin aIIb upregulation may competitively inhibit binding offibronectin to common pro-fibrotic integrins. Further affinity assaysare ongoing to understand potential mechanisms behindendostatin-integrin-FN interactions. The inventors also found enhancedexpression of HGF after Fc-endostatin and mP1. HGF has been recentlyidentified to elicit putative anti-fibrotic effects [Crestani, B., etal., Hepatocyte growth factor and lung fibrosis. Proc Am Thorac Soc,2012. 9(3): p. 158-63; Phin, S., et al., Imbalance in the pro-hepatocytegrowth factor activation system in bleomycin-induced lung fibrosis inmice. Am J Respir Cell Mol Biol, 2010. 42(3): p. 286-93].

The most striking data the inventors could provide so far is that theN-terminal zinc binding region of endostatin known to be chieflyinvolved in its anti-angiogenic effects [Tjin, R. M., et al., A27-amino-acid synthetic peptide corresponding to the NH2-terminalzinc-binding domain of endostatin is responsible for its antitumoractivity. Cancer Research, 2005. 65(9): p. 3656-3663] is also relevantfor the anti-fibrotic effect elicited by this endogenous protein. Thisis in clear contrast to recently published data postulating ananti-fibrotic effect of the C-terminal domain of endostatin [Yamaguchi,Y., et al., A peptide derived from endostatin ameliorates organfibrosis. Sci Transl Med, 2012. 4(136): p. 136ra71]. In the radiationinduced lung fibrosis model used by the present inventors, theC-terminal peptide was not effective to improve most investigatedparameter of fibrosis development. Together, the data of the presentinventors indicate an important role for the N-terminus sequence as wellas dimerization of endostatin underlying its anti-fibrotic effect in theRILF model.

Example 11: Binding Properties of Oligomeric Endostatin

The present inventors have previously shown that the anti-fibroticeffect of endostatin could most conceivably not be confined to itsC-terminal fragment as proposed by Yamaguchi et al., 2012, loc. cit. Acloser look at the endostatin C-terminus, the E4 peptide containingarea, shows no obvious structural feature linking this fragment withpotential protein interaction partners that could provide a mechanisticexplanation for the postulated anti-fibrotic effect of the molecule.Another explanation for the lack of E4 activity might be that incontrast to their acute murine fibrosis models, the present inventorsutilized a radiation induced lung fibrosis model, where fibrosisdevelopment follows a slow (over 24 weeks after irradiation) and chronickinetic more closely resembling the pathophysiology in humans.

The present inventors further showed that the N-terminal zinc bindingfragment elicit moderate anti-fibrotic activity. However, the mostefficient attenuation of lung fibrosis was found when a syntheticendostatin dimer (Fc-endostatin) was utilized. Fc-endostatin (FcE)consists of two Fc chains (connected by disulfide bonds), extended totwo molecules of endostatin each linked to a single Fc chain. Therefore,the two adjacent endostatin molecules become a dimer as a result of theFc dimer.

The present inventors previously have shown that the physiologicmolecule circulating in the human blood is endostatin precursor NC1fragment of collagen 18 which is an oligomeric endostatin molecule withthree endostatin domains (endostatin trimer). Moreover, the presentinventors showed that mixing Fc-endostatin with platelets lysate,fibronectin (FN) was immune-precipitated without need for additionalantibody to facilitate their interactions. The data of the presentinventors later made it clear that binding of FN is unique to oligomericendostatin (dimer or trimeric NC1) and is not shared by endostatinmonomer which is considered so far as the key anti-angiogenic moleculederived from collagen 18 (FIG. 9 ). Of note, fibronectin is a centralmolecule in development of tissue fibrosis. Hence hypothesis by thepresent inventors is that the beneficial anti-fibrotic effect of theoligomeric endostatin is at least in part mediated by its property tobind FN, and this distinguishes the NC1 or endostatin oligomers frommonomers or fragments thereof (N-terminal, middle or C-terminalfragments).

In the present inventors' view, endostatin is an end-degradation productof NC1. They present here new data further demonstrating that thebinding properties of endostatin dimer and NC1 trimer are quite distinctfrom endostatin monomer in terms of relevant protein interactionpartners. In other words, oligomerization properties of endostatin playan important role in its binding to key players of tissue remodelingwith high impact for exploration of its anti-fibrotic and anti-cancereffects.

A novel finding of the present inventors is the binding of oligomericendostatin to the vascular endothelial growth factor (VEGF), a pivotalmolecule in a number of so called VEGF-related diseases encompassing abroad range of pathophysiologic conditions from wet-macular degenerationto cancer and fibrosis. Indeed, Nindetanib which was recently approvedfor treatment of pulmonary fibrosis is a potent inhibitor of PDGF andVEGF signaling. In contrast to endostatin dimer and NC1, VEGF does notbind to endostatin monomer (FIG. 10 ).

Crystallography of endostatin had previously demonstrated that thisprotein was a dimer each binding an atom of zinc (Ding, Y. H., K.Javaherian, K. M. Lo, et al. 1998. Zinc-dependent dimers observed incrystals of human endostatin. Proc. Natl. Acad. Sci. U.S.A. 95,10443-10448). Interestingly, the N-terminal zinc binding domain ofendostatin resembles that of MMPs (matrix metalloproteinases); importantplayers in remodeling of extracellular matrix, in development offibrosis, cancer progression and metastasis. Here, the present inventorsdemonstrate that indeed endostatin dimer and NC1 trimer bind to MMP-2and MMP-9; a property not shared by endostatin monomer (FIG. 11 ). Thisfinding opens a new avenue for pursuing biological properties ofoligomeric endostatin, either by synthetic design, e.g., dimerizationvia Fc or other alternatives to generate and improve a drug mimickingthe endostatin precursor molecule NC1.

Based on the studies of the present inventors of crystal structure ofendostatin dimer, they recognized that amino acids glutamine at position7 from N-terminus is closely adjacent to the same amino acid in thesecond chain. They replaced Q (Gln) by C (Cys) in this positionpredicting that an artificial endostatin dimer would result, covalentlyattached by a disulfide bond. Their prediction turned out to be correct.The new artificial dimer was expressed in Fc-endostatin vector asbefore. However, both Fc and endostatin were separately dimerized bytheir corresponding disulfide bonds. Enterokinase digestion of thisrecombinant protein resulted in an Fc dimer and an endostatin dimerwhich were purified on an S-200 Sephadex. The term “endostatin dimer”(ES dimer) employed in all binding assays presented in this Example 11refers to this purified molecule.

Another convincing evidence in support of their hypothesis that bindingproperties presented here are observed with oligomeric endostatin only,is shown in FIG. 12 . An enterokinase binding site is engineered betweenFc and endostatin in Fc-endostatin. Digestion of this molecule withenterokinase (EK) resulted in Fc dimer and two endostatin monomers.Without any additional modifications, mixture of Fc and endostatindisplayed distinct properties from intact Fc-endostatin.

Together, the present inventors show here additional data unravelingnovel bindings partners for oligomeric but not monomeric endostatin withpivotal roles in development of organ fibrosis. The unique property ofoligomeric endostatin to target these molecules provides a plausibleexplanation for the superior anti-fibrotic activity of NC1 or NC1- likeoligomeric endostatins (e.g. Fc-ES) over the monomeric end degradationproduct or even peptide fragments thereof (mP1 or E4) tested by thepresent inventors in the murine lung fibrosis model. The novel findingsby the present inventors opens a new avenue for pursuing the developmentof NC1 or NC1-mimetics consisting of oligomeric endostatin (at least adimer) for the treatment of not only fibrosis-related diseases, but alsoVEGF-related diseases, MMP-dependent diseases, and the modulation offibronectin function.

The reagents which have been used in Example 11 are listed, in thefollowing:

-   -   Corning , 96 well EIA/RIA High Bind, polystyrene, flat bottom,        clear, non-sterile #3590    -   BSA (Sigma Aldrich #A7030 IgG free)    -   recombinant hMMP-2 (R&D #902-MP-010) >1 μg/ml in PBS    -   recombinant hMMP-9 (R&D #911-MP-010) >1 μg/ml in PBS    -   R7012 a.p. (anti-endostatin antibody)    -   ABTS (Rockland #ABTS-100)    -   Peroxidase Conjugated Affini Pure Goat anti-rabbit IgG (H+L)        (Jackson Immuno Research # 111-035-003 2.0 ml)    -   hFN (R&D #1918FN-02M)    -   Recombinant Human VEGF165 (R&D Systems a biotechne brand        #293-VE-010/CF)

Proteingel:

-   -   Page Ruler Plus Prestained Protein Ladder (Thermo Scientific        #26619)    -   SDS page 4-20% : Mini-Protean TGX Precast Protein Gels (BioRad        #4561094)    -   Enterokinase, Light Chain, Porcine (GenScript #Z01003

1-18. (canceled)
 19. A method for treating, ameliorating or preventing amatrix metalloproteinase (MMP)-related disease in a patient in needthereof, the method comprising administering to the patient atherapeutically effective dose of a protein oligomer comprising (i) atleast two NC-1 monomers of collagen 18 or (ii) at least two endostatindomains of collagen 18 or (iii) at least two N-terminal peptides of thecollagen 18 endostatin domain, thereby, treating, ameliorating orpreventing said matrix metalloproteinase (MMP)-related disease in thepatient, wherein the NC-1 monomer of human collagen 18 comprises aheterologous oligomerization domain comprising an Fc domain fromknobs-into-holes (KiH)-engineered human IgG, a hinge region and/or anendostatin domain or fragments of said endostatin domain and,optionally, a recombinant protease cleavage site within the hingeregion.
 20. The method of claim 19, wherein the Fc domain is fromknobs-into-holes (KiH)-engineered human IgG1.
 21. The method of claim20, wherein the Fc domain comprises the amino acid sequence of SEQ IDNos. 25, 26, 28 and/or
 30. 22. The method of claim 19, wherein saidheterologous oligomerization domain further comprises an artificialoligomerization domain.
 23. The method of claim 19, wherein said proteinoligomer further comprises a native oligomerization domain.
 24. Themethod of claim 19, wherein the endostatin domain of collagen 18 isselected from an amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 19or the N-terminal peptide of the collagen 18 endostatin domain isselected from an amino acid sequence from amino acid residue 1 to 132 ofSEQ ID NO: 18 or SEQ ID NO:
 19. 25. The method of claim 19, wherein saidprotein oligomer further comprises an RGD motif and/or PHSRN motif ofFibronectin, in the NC-1 monomer of collagen 18, the endostatin domainof collagen 18 or the N-terminal peptide of the collagen 18 endostatindomain.
 26. The method of claim 19, wherein said protein oligomer bindsto Fibronectin, VEGF, MMP-2 and/or MMP-9.
 27. The method of claim 19,wherein said protein oligomer further comprises angiostatin,thrombospondin, anti-PD-1/PD-L1 antibodies or another therapy employedfor the MMP-related disease.
 28. The method of claim 19, wherein thematrix metalloproteinase (MMP)-related disease is selected from thegroup consisting of a benign and malignant disease where MMP activationcontributes to the pathophysiology, overt immune reaction in autoimmunediseases, and chronic inflammatory diseases.
 29. The method of claim 19,wherein the protein oligomer is administered at a concentration of 0.1-1mg/kg/day.
 30. The method of claim 19, wherein the protein oligomer isadministered intravenously, intracranial/intrathecal, intravitreal,subcutaneously or intraperitoneally.
 31. The method of claim 19, whereinthe protein oligomer has one or more biological activities selected fromanti-fibrotic activity, anti-angiogenic activity,anti-invasive/anti-metastatic activity, reducing vascular permeabilityactivity, anti-inflammatory, and/or anti-tumorigenic activity.
 32. Themethod of claim 22, wherein the artificial oligomerization domaincontains a single mutation at position 7 of the endostatin domain ofcollagen 18 in which glutamine is replaced by cysteine.
 33. The methodof claim 23, wherein the native oligomerization domain is a non-triplehelical trimerization domain of collagen
 18. 34. A method for detectingand/or diagnosing a patient suspected of suffering from a matrixmetalloproteinase (MMP)-related disease, the method comprisingadministering to said patient a protein oligomer comprising (i) at leasttwo NC-1 monomers of collagen 18, (ii) at least two endostatin domainsof collagen 18 or (iii) at least two N-terminal peptides of the collagen18 endostatin domain, wherein the NC-1 monomers of human collagen 18,the endostatin domain of collagen 18 or the N-terminal peptides of thecollagen 18 endostatin domain are labeled with radioisotopes,radionuclides binding to chelates, fluorescent proteins or other labels,thereby, detecting and/or diagnosing matrix metalloproteinase(MMP)-related disease in the patient.